Medical Biochemistry PDF

MEDICAL BIOCHEMISTRY THIS PAGE IS BLANK Copyright © 2006, 2002 New Age International (P) Ltd., Publishers Published by New Age International (P) Ltd., Publishers All rights reserved. No part of this ebook may be reproduced in any form, by photostat, microfilm, xerography, or any other means,...

MEDICAL BIOCHEMISTRY THIS PAGE IS BLANK Copyright © 2006, 2002 New Age International (P) Ltd., Publishers Published by New Age International (P) Ltd., Publishers All rights reserved. No part of this ebook may be reproduced in any form, by photostat, microfilm, xerography, or any other means, or incorporated into any information retrieval system, electronic or mechanical, without the written permission of the publisher. All inquiries should be emailed to [email protected] ISBN : 978-81-224-2300-6 PUBLISHING FOR ONE WORLD NEW AGE INTERNATIONAL (P) LIMITED, PUBLISHERS 4835/24, Ansari Road, Daryaganj, New Delhi - 110002 Visit us at www.newagepublishers.com  To m myy Elder daughter Late Nallur Nallurii Kiranmayi Chowdary THIS PAGE IS BLANK  PREFACE TO THE SECOND EDITION I attempted to provide essential information on molecular basis of health and disease that is mainly related to life of surviving cell(s) in the first edition of the book. However life cycle of cell(s) includes cell(s) birth and cell(s) death apart from survival. For the last couple of years these frontier areas are advancing rapidly which is viewed by many as good sign for development of : (a) new therapy or therapeutics for cancer (b) immortalized cells. The latter fuels growth of biotechnology and pharmaceutical industries also. Hence, in the second edition two chapters—1. Biochemistry of cell cycle (cell birth) 2. Biochemistry of apoptosis (cell death) are added. As living organisms evolved from simple unicellular to highly complex multicellular mammals, several new systems and organs were developed. For example, blood which acts as vehicle or communication between various locations of body, immune system which protects body from intruders or foreign organisms. Parkinsonism, psychosis, depression, Schizophrenia, loss of taste and olfaction are due to disturbances in nervous, taste and olfactory systems. Various organs present in body perform several organ specific functions which are essential for life. If functions of these organs are disturbed, diseases in which may culminate in death. So, in this edition biochemistry of blood including immune response in Chapter-32; molecular and cellular mechanism of learning, memory, behaviour, taste and olfactory in biochemical communications Chapter; tests, procedures that are done in hospital biochemistry laboratory to assess functions of liver, kidney in Chapter-33 and thyroid in Chapter-29 are detailed. Depending on disease a particular constituent of blood is either elevated or lowered. Diagnosis and prognosis of disease usually involves detection and measurement of various blood constituents in hospital biochemistry laboratory. Therefore advanced techniques like high performance liquid chromatography (HPLC), affinity chromatography, and general techniques like centrifugation and dialysis, instruments from spectrophotometer to auto analyzer and methods used for detection and quantitation of blood constituents like carbohydrates, proteins, lipids, nucleic acids, enzymes, electrolytes etc., in health and disease are detailed in Chapter-34. Humans and other mammals are able to remove waste products, toxins, foreign compounds from blood and organs in the form of urine. In disease, composition of urine varies from that of healthy state. So, detection, quantitation of various constituents of urine is carried out in hospital biochemistry laboratory to confirm diagnosis of diseases. In Chapter- (vii)  (viii) 34, methods for detection and estimation of urine constituent under normal and diseased conditions are also detailed. Most striking feature in this edition of the book is inclusion of biochemical aspects of diseases or disease causing organisms common to tropical countries like malaria, tuberculosis, peptic ulcer or gastritis, pneumonia, leishmaniasis, giardiasis, trypanosomiasis etc. Since most of the organisms are developing resistance to the existing drugs, there is need for development of new drugs which requires thorough biochemical knowledge of these diseases as well as disease causing organisms. Apart from adding new chapters, all existing chapters have been updated by adding new subject matter. References of each chapter updated by including reviews, books, research articles etc. Further, number of unsolved problems have been increased in most of the chapters. I hope this edition will be well received by teachers and students of various medical, dental, pharmacy, biotechnology, physiotherapy, medical laboratory technology, biomedical engineering, life sciences under graduate and post graduate courses, Suggestions or comments from teachers and students are welcome. I am grateful to Sri. R.K. Gupta, Chairman; Sri Saumya Gupta, Managing Director of New Age International, New Delhi, for publishing second edition. N. MALLIKARJUNA RAO  PREFACE TO THE FIRST EDITION This book explains the fundamentals of biochemical (molecular) bases of health and disease. Hence it meets medical and allied health sciences student’s needs. As a teacher of medical, dental, pharmacy, biomedical engineering and science students for the last two decades, I know the problems faced by students in mastering (conceptualizing) the subject within a limited time. Most of these students need a book for their routine day-to-day study which contains only the necessary information in a simple and concise way. Therefore, this book is written in simple language in such a way that a student with very little chemistry or biology background can easily follow the various aspects of biochemistry that are presented. This book is also useful for those who are specializing in biochemistry (M.Sc. or M.D.) because advances in frontier areas of biochemistry are presented in a systemic way. Of course advances in other areas that are relevant to medical students are also included to a limited extent. An interesting feature of this book is that the medical and biological importance of each chapter is highlighted in simple numbered statements. Further, in some chapters, diseases, drugs (treatments) or toxins of particular subject matter are described under medical importance heads. Further, each chapter’s text is designed to facilitate easy flow of information in an interesting, thought provoking and logical manner. Exercises (cases) given at the end of each chapter help in mastering of the subject by student and utilization of biochemical principles by the student in solving health problems. To enthusiastic students, references given at the end of each chapter provide additional information. There are 29 chapters in the book. First six chapters deal with the composition, structure, function and life cycle of cells and the goal of biochemistry; occurrence, chemistry, structure and functions of biomolecules like amino acids, peptides, proteins, enzymes, carbohydrates and lipids. This is then followed by chapters 7 and 8 that deal with membrane structure, various transporters that move biomolecules across membrane and disintegration of complex molecules of food and absorption of resulting products, respectively. Chapters 9-12 deal with the production and utilization of energy in various pathways of carbohydrate, lipid and aminoacid metabolisms. Regulatory mechanisms of some of the important pathways are also outlined. Further synthesis of biologically (medically) important compounds including non-essential amino acids is detailed. In chapter 11 the ultimate way of producing energy from all energy yielding compounds in the respiratory chain is described. Changes in the flow of metabolites into various pathways of carbohydrate, lipid and protein metabolism that occur among tissues in well fed state, diabetes and starvation are described in chapter 13. Fundamentals of molecular biology i.e., occurrence, chemistry, structure, functions, metabolism of nucleotides, nucleic acids and control of gene expression as well as applied molecular biology i.e., recombinant DNA technology are detailed in chapters 14-20. Biomedical (ix)  (x) (chemical) aspects of two major health problems of the 20th century—cancer and AIDS are briefed in chapter 21. In chapter 22 occurrence, chemistry, structure, functions and metabolism of porphyrins and hemoglobin are described. Clinically related topics like vitamins, minerals, macro nutrients, energy, nutraceuticals of food, electrolytes, acid-base balance and detoxification are described in chapters 23-27. Chemistry, production, detection and uses of isotopes in biochemistry and medicine are detailed in chapter 28. Chapter 29 deals with mechanisms of communication between cells. I hope both teachers and students of Biochemistry at undergraduate and postgraduate levels use this book extensively and their suggestions to improve the book further are most welcome. I express my sincere thanks to New Age International, Publishers for publishing the book. N. MALLIKARJUNA RAO  CONTENTS I'"fffff to 1M S«t.md &Ii/ion «>ii) I'"ff{oct to flu Fi,.., Edition (u)..,. Coli , AminoAcids and Peptides 3. Proteins ,." ElUymes alogy...'" ". Cancer and Aids.90 22. Porphyrin and HaemOjlobi n Metabolism 23. Vitamirul "" ". Minerala '" ,n " (:ai) Energy, Nutrients. Medie;nft and 'Ibxins of Food 26. Wate_r. Electrolytes and Acid Bue Balanoo.. 629 , GA). Hence, the conversion of A to B takes place when energy is supplied and reaction occurs with free energy increase. Endergonic reactions These reactions occur when energy is supplied. These reactions consume energy in biologi- cal systems. Determination of ∆G The free energy change of a chemical reaction A → B is determined by equation. [B] ∆G = ∆G1 + RT ln [A] ∆G1 = standard free energy change when concentrations of A and B are 1M T = absolute temperature, R = Gas constant At equilibrium ∆G = 0 ln[B] Then ∆G1 = −RT [A] [B] = –RT In Keq (Equilibrium constant Keq = ) [A] For biochemical reactions, ∆G0| is used instead of ∆G1. ∆G0| is the standard free energy change of a reactions at pH 7.0. High energy compounds The hydrolysis of these compounds is accompanied by release of large amount of free energy. Since ATP is a high energy compound, the energy released during transfer of electrons from reduced coenzymes to O2 is conserved in the form of ATP. The energy released when an high energy compound is hydrolyzed is not due to bond that is hydrolyzed. It is due to large difference in the free energy content of reactant and product. 272 Medical Biochemistry ∆G01 for the hydrolysis of ATP is given below ATP → ADP + Pi ∆G0| = –7.3 Kcal/mol (–30 KJ/mole) ADP is also energy rich compound because ∆G01 for ADP hydrolysis is –7.3 Kcal/mol. By convention, high energy bond is shown with ~ (squiggle) symbol. So, the ATP is written as High energy bond Adenine-Ribose- P ~ P ~ P ∆G for hydrolysis of other compounds is less. For example, when glucose-6-phosphate 01 is hydrolysed only 3.3 Kcal/mol of energy is released (∆G01 = -3.3 Kcal/mol). Significance of ATP 1. It is involved in the transfer of energy in the cells. It is often called as energy currency of the cell. 2. In the cells, the energy released in an exergonic reaction is used to form ATP and energy required for an endergonic reactions is supplied by hydrolysing ATP. Therefore, in biological systems ATP serve as link between the exergonic and endergonic reactions (Fig. 11.7). 3. Energy of ATP hydrolysis is also used for muscle contraction, transport of ions and molecules across cell membrane, motility of sperm cells etc. Other nucleoside triphosphates like GTP, UTP, CTP, TTP, dATP, dGTP and dTTP are also high energy compounds. The electronic structure of these compounds is responsible for the release of large free energy on hydrolysis. Fig. 11.7 Schematic diagram showing ATP link between exergonic and endergonic reactions Biological Oxidation and Respiratory Chain 273 Other high energy compounds are Phospho creatine It is present in the skeletal muscle. It is involved in the transfer and storage of energy in muscle. The free energy of hydrolysis of phosphocreatine is 10.3 Kcal/mol (∆G0| = –10.3 Kcal/ mol). Thioesters They are formed from the condensation of coenzyme A, a thiol with carboxylic acids. They are also high energy compounds. For example, hydrolysis of acetyl-CoA to acetic acid and water is accomanied by release of 7.5 Kcal/mol of energy (Figure 11.8a). Enolphosphates They are esters of enol with phosphoric acids. Phosphoenol pyruvate is an example for enol phosphate ester. The free energy released on hydrolysis of this high energy compound is –14.8 Kcal/mol (Figure 11.8b). Acyl phosphates They are mixed anhydrides. The two acids involved in the mixed anhydride formation are carboxylic acid and phosphoric acid. An example for a high energy mixed anhydride is 1, 3- bisphosphoglycerate. Hydrolysis of 1, 3-bisphospholgycerate is accompanied release of –11.8 Kcal/mol of energy (Figure 11.8c). O O A CH3 C ~S CoA CH3 C OH C oA S H A cetyl-C o A H 2O A cetic a cid co enzym e A ∆G O | = – 7.5 K ca l/m ol COOH COOH B C O~ P C O CH2 H 2O CH3 P h osp ho en ol P yru va te p yru vate (P E P ) ∆G O | = –1 4.8 K ca l/m o l O O C O ~P C OH C CH OH H C OH H 2O CH2 O P CH2 O P 1 ,3– bisph osph o glycerate 3 -P ho sph og lycera te ∆G O | = –11.8 K cal/m ol Fig. 11.8 (a) Hydrolysis of Acetyl-CoA. (b) Hydrolysis of PEP (c) Hydrolysis of 1, 3-bisphosphoglycerate Redox potential It is an electrochemical concept related to redox reactions. 274 Medical Biochemistry Redox reactions The oxidation-reduction reactions are called as redox reactions. The oxidant (acceptor) and reductant (donor) of a redox reaction are known as redox pair or redox couple. For example, NAD (oxidized form)/NADH (reduced form) constitutes a redox couple. Redox potential It is defined as electro motive force (e.m.f.) of a redox pair when oxidant and reductant are present in 1M concentration. The symbol used to indicate redox potential is E01 and units are volts. It indicates the tendency of a redox pair to gain or give electron. The redox potentials of biologically important redox pairs are measured at pH 7.0 by taking hydrogen electrode as standard (reference). Redox potentials of some redox pairs Redox pair Redox potential (E01) in volts O2/H2O +0.82 Cytochrome c +0.25 FAD/FADH2 –0.18 + + NAD /NADH+H –0.32 α-ketoglutarate/isocitrate –0.38 Acetyl-CoA/Pyruvate –0.48 If the redox potential E01 of redox pair is more negative, then it always undergo oxida- tion or tend to loose electrons. Likewise, if E01 is positive for a redox pair then, it accepts electrons or undergo reduction. For example, take isocitrate/α-ketoglutarate with E01 of – 0.38 v and NAD+/NADH+H+ with E01 of –0.32 v, the electrons always pass from former redox pair to latter redox pair. Electron transfer and free energy When electrons flow from electronegative redox pair towards electropositive redox pair free energy is liberated. The amount of free energy liberated when electrons move from one redox pair to another is given by the equation. ∆G0| = −nf∆E1 0 ∆G0| = standard free energy change in calories n = number of electrons transferred f = faraday (23.6 Kcal) ∆E10 = Difference between the redox potentials of elec- tron donor and acceptor The negative sign on the right hand side of the equation indicates release of free energy into surroundings. The equation also indicates that the amount of free energy liberated depends on difference of the redox potential between two redox pairs. So, when electrons flow from NAD to O2 large amount of free energy is released because of the more redox potential difference between two redox pairs. By sustituting redox potentials of NAD/NADH2 and O2/H2O in the above equation, we get Biological Oxidation and Respiratory Chain 275 ∆G0| = –2 × 23.6 × (–0.32 –0.82) = –56.2 Kcal Since only 7.5 Kcal of energy is required for the formation of one molecule of ATP from ADP and Pi, during the transfer of electrons from NAD to O2, three ATP are generated and remaining energy is released as heat. In addition, this equation can be used to know redox potential difference required for ATP formation in the respiratory chain. Approximately 0.15 volts of redox potential difference is required for one ATP formation. ELECTRON TRANSPORT CHAIN (ETC) OR RESPIRATORY CHAIN It is present in inner mitochondrial membrane. Electron transport chain consist of various electron transport or electron carrier molecules. The electron transport molecules are ar- ranged in a sequence. They carry or transfer electrons from reduced coenzymes like NADH, FADH2 to final electron acceptor O2. The electron transport molecules are called as compo- nents of respiratory chain. Some of them are proteins and non-protein carriers also present. The position of a particular component in the respiratory chain depends on its redox potential. The components of respiratory chain are arranged in the order of increasing redox potential. Starting components have negative redox potential and terminal components have positive redox potential. Therefore, in the respiratory chain, electrons flow from negative to positive (Figure 11.9). P yru va te S u ccina te α-K e to glutara te G lycero l-3 -p ho sp h ate FA D FA D (– 0.1 8 V ) NAD FM N Co Q C yt b C yt c 1 C yt c C yt a C yt a 3 O2 (– 0.3 2 V ) (– 0.1 2 V ) (+ 0.0 4 V ) (+ 0.0 7 V ) (+ 0.2 3 V ) (+ 0.2 5 V ) (+ 0.2 9 V ) (+ 0.5 5 V ) (+ 0.8 2 V ) M alate g lu tam a te Fig. 11.9 Electron flow in the respiratory chain. Redox potential of each component of electron transport chain is given in parenthesis In the respiratory chain, electrons flow from NAD to cytochromes via CoQ and then from cytochromes to molecular O2. CoQ also collects electrons from FAD. The transfer of electrons from substrates to NAD and FAD is catalyzed by dehydrogenases. At the electronegative end of respiratory chain, NAD linked deydrogenases like malate and glutamate dehydrogenases catalyze transfer of electrons from substrate to NAD directly but electrons from substrates like pyruvate, and α-ketoglutarate are transferred via FAD (Figure 11.9). In contrast some FAD linked dehydrogenases like succinate and glycerol-3-phosphate dehydrogenases catalyze transfer of electrons from substrate to CoQ of respiratory chain because their redox potentials are more positive. Many components of respiratory chain are present as complexes rather than single entities. On NAD, CoQ and cytochrome c are present as individual components rest of the components of respiratory chain are present as complexes. The repiratory chain consist of four complexes and three mobile carriers. The complexes are complex I, II, III and VI and 276 Medical Biochemistry mobile carriers are NAD, CoQ, cytochrome c and O2. All the complexes are integral mem- brane proteins. Each complex is involved in oxidation and each complex accepts electrons from mobile electron carrier and pass electrons to another mobile electron carrier (Figure 11.10). Complex I is NADH-CoQ reductase or NADH dehydrogenase. FMN and Fe:S clusters are prosthetic groups of this enzyme. Electrons collected by NAD are transferred to CoQ by NADH-CoQ reductase via FMN and Fe:S cluser. So NAD is oxidized. Complex II is the succinate-CoQ reductase. It also contains FAD and iron sulfur clusters. Complex II transfers electrons from succinate and glycerol-3-phosphate to CoQ via FAD and Fe:S. Complex III is CoQ-cyt c reductase or cytochrome reductase. It also contains Fe:S cluster. Complex III transfers electrons from CoQ to cytochrome c via cyt b, c, and Fe:S cluster. As a result of this CoQ get oxidized. Complex IV is cytochrome oxidase. It transfers the electrons from cyt c to final electron acceptor O2 so cyt c is oxidized (Figure 11.10). S u ccina te C o m FA D p S u ccina te- l C o Q R e du ctase e Fe : S x II N A D H –C o Q C yto ch rom e R e du ctase C o Q -C yt c re du ctase o xida se O2 NAD FM N Fe :S Co Q C yt b Fe :S C yt c 1 C yt c C yt a a 3 H 2O C o m p le x I C o m p le x III C o m p le x IV Fig. 11.10 Complexes and mobile carriers of respiratory chain Oxidative phosphorylation During the transfer of electrons in the respiratory chain, energy is released because elec- trons flow from electronegative NAD to electropositive O2. The energy so released is used for the formation of ATP from ADP and Pi. The generation or synthesis of ATP from ADP and phosphate (Pi) while electrons flow in the respiratory chain is called as oxidative phosphorylation. This process accounts for over 85% of high energy phosphates or ATP produced in the body. Oxidative phosphorylation is the combination of oxidation and phosphorylation. The two processes are coupled to each other and in normal cells one does not usually occur without the other. The word oxidation is used because the transfer of electrons from substrate to oxygen causes oxidation of substrate or when mobile carrier is oxidized then only electrons flow occurs in the respiratory chain. ATP synthesis Formation of ATP from ADP and phosphate using energy released when electrons flow in the respiratory chain is catalysed by membrane bound enzyme known as ATP synthase or F0 F1 ATPase. It is often referred as another complex V of respiratory chain. It is an integral membrane protein of inner mitocondrial membrane. ATP synthase is oriented vectorially in the inner mitochondrial membrane. It extends from outside of inner mitochondrial mem- brane to matrix of mitochondria. It is present in the knob-like structure present on the cristae of inner mitochondrial membrane. Biological Oxidation and Respiratory Chain 277 ATP synthase consist of two subunits. They are F0 and F1 subunits. The spherical part or head of knob is F1 subunit. It is made up of five polypeptide chains (Figure 11.11a). This subunit has catalytic activity. It catalyzes the hydrolysis of ATP in vitro. The base of the know is F0 subunit of ATP synthase. It is embedded in the membrane. It is channel for proton movement (Figure 11.11a). When protons move through F0 subunit from outside to inside of inner mitochondrial membrane F1 subunit catalyzes the formation of ATP from ADP and Pi using free energy released. The stalk of knob also consist of several proteins. Antibiotic oligomycin binds to stalk and inhibits oxidative phosphorylation. Sites of phosphorylation or ATP synthesis in the respiratory chain ATP synthesis takes place only at specific points of respiratory chain like any energy yielding degradative pathways like glycolysis, β-oxidation etc. ATP synthesis in respiratory chain requires a redox potential difference of approximately 0.15 volts. Each complex of respiratory chain generates 0.15 v redox potential difference while electrons flow from NAD to O2. ATP generation is associated with three complexes of electron transport chain and it is always coupled to the flow of electrons through the complexes. The three complexes that generate ATP from ADP and Pi are complex I, complex III, and complex IV (Figure 11.11b). O ut In ne r sid e m ito cho nd ia l (a ) m em b ran e In side ATP H+ H+ ADP + Pi F 0 su bu nit S ta lk F 1 su bu nit (b ) NADH–Co Q C yto ch rom e re du ctase C o Q -C yt c re du ctase o xida se NAD FM N Fe : s Co Q C yt b Fe : s C yt c 1 C yt c C yt a a 3 O2 AT P AT P AT P s yn th a se s yn th a se s yn th a se ADP + Pi ATP ADP + Pi ATP ADP + Pi ATP Fig. 11.11 (a) Structure of ATP synthase (Complex V) (b) Sites of ATP formation in respiratory chain P:O ratio When electrons flow in the respiratory chain from NAD to O2 formation of ATP occurs and at the same time O2 is reduced to H2O. The relationship between ATP generation and oxygen consumption is expressed as P:O ratio. It is defined as ratio of number of ATP synthesized per atom of oxygen consumed when electrons flow in the respiratory chain from substrate to O2. When a substrate like malate is oxidized by NAD linked malate dehydrogenase NADH is produced. Oxidation of this NADH in respiratory chain is accompanied by forma- tion of 3 ATP molecules and one oxygen atom is reduced of water and the P:O ratio is 3. 278 Medical Biochemistry So, oxidation of a substrate by NAD linked dehydrogenase generates 3 ATP molecules. Likewise, oxidation of a substrate by Flavoprotein (FAD) dehydrogenase generates 2 ATP molecules per atom of oxygen consumed, i.e., P:O ratio is 2. The difference in the P:O ratio is due to by passing the complex I of the respiratory chain. Synthesis of ATP in respiratory chain when a substrate is oxidized is known as oxidative phosphorylation at respiratory chain level. Substrate level phosphorylation It is another process for production of high energy compounds like ATP and GTP. Enol phosphate or thioesters are produced in metabolic particular degradative pathways and they are subsequently utilized to generate ATP. For example in glycolysis phosphoenol pyruvate is produced and it is subsequently used to generate ATP. Inhibitors of respiratory Chain Several drugs, poisons and toxins work by inhibiting activities of respiratory chain com- plexes. They block oxidative phosphorylation or ATP synthesis or flow of electrons in the respiratory chain. Some compounds uncouple the oxidation and phosphorylation. Compounds that inhibit oxidative phosphorylation They act mainly at the sites of ATP synthesis. (a) Compounds which act at NADH-CoQ reductase or complex I These compounds block ATP synthesis at site I. 1. Amytal (barbiturate) used as sedative inhibit NADH-CoQ reductase. So, electron flow is blocked ATP synthesis does not occur. 2. Rotenone a fish poison inhibits complex I activity. It blocks flow of electrons from Fe:S clusters. However, it is non-toxic to humans. It is a plant product and used as insecticide also. 3. Piericidin, an antibiotic also blocks activity of complex I. (b) Compounds which act at complex II or II site of ATP synthesis These compounds inhibit the formation of ATP at second site. 1. Antimycin A: An antibiotic blocks electron transport at site II by inhibiting cytochrome reductase. 2. BAL (British anti lewisite): It is used as therapeutic agent in the cases of arsenic poisoning. It inhibits activity of cytochrome reductase. (c) Compounds which act at site III by inhibiting activity of cytochrome oxidase. 1. Cyanide (CN) A powerful poison that inhibit cytochrome oxidase by combining with cytochrome a3. Cyanide may arise from cyanogenic substance. 2. Carbon monoxide (CO) It inhibits activity of cytochrome oxidase. Carbon monoxide is a pollutant present in automobile exhaust. 3. Hydrogen sulfide (H2S) It inhibits cytochrome oxidase. H2S toxicity occurs during oil drilling operations. It is toxic as cyanide. It is a part of natural gas. 4. Azide Sodium azide also inhibits cytochrome oxidase activity. Biological Oxidation and Respiratory Chain 279 Other inhibitors of oxidative phosphorylation 1. Carboxin It inhibits oxidative phosphorylation by blocking the transfer of electrons from succinate to CoQ. 2. Atractyloside It inhibits oxidative phosphorylation by blocking movement of ATP and ADP across inner mitochondrial membrane. 3. Oligomycin It interacts with stalk of knob structure and completely blocks oxidation and phosphorylation. 4. Rutamycin It blocks phosphorylation without uncoupling. Uncouplers These compounds dissociates or uncouples oxidation in respiratory chain from phosphorylation. So, the oxidation takes place without ATP synthesis. Examples: (1) 2, 4-dinitrophenol (2) Dinitrocresol (3) Salicylanilides (4) Pentachlorophenol (5) CCCP (Carbonylcyanide chloromethoxy phenyl hydrazone). (6) FCCP (Carbanoyl cyanide p. trifluoromethoxy phenyl hydrazone). Regulation of oxidative phosphorylation Oxidative phosphorylation in the respiratory chain is subjected to regulation like any meta- bolic pathway. The rate of oxidative phosphorylation depends on availability of substrates like ADP, P i, NADH, FADH 2 , and O 2. When cell has enough ATP the oxidative phosphorylation occurs at lower rate because of non availability of ADP. When the cell is deficient in ATP, ADP availability is more so rate of oxidative phosphorylation is more. The rate of oxidative phosphorylation also depends on the availability of Pi. Therefore, the energy generation in the mitochondria is perfectly tuned to energy demand. The dependence of oxidative phosphorylation on the availability of ADP is known as respiratory control. Mechanism of oxidative phosphorylation Three models have been proposed to explain ATP synthesis during the transfer of electrons in the respiratory chain. They are 1. The chemical coupling model According to this model, when electrons are transferred in the respiratory chain, an high energy intermediate is formed. The hydrolysis of this high energy compound is accompanied by the formation of ATP. No such high energy intermediate has been found so far. 2. The conformational coupling model This model proposes the existence of two conformational states to the inner mitochondrial membrane components. The energy released when electrons flow in the respiratory chain causes conformational change in these components and converts low energy molecules to high energy form. When they return to normal low energy state from high energy state the energy released is used for ATP synthesis. Due to lack of experimental support this model has not been accepted. 3. The chemiosmotic coupling model According to this model, when electrons flow in the respiratory chain, protons (H+) are pumped from matrix of mitochondria to outside of inner mitochondrial membrane. As a 280 Medical Biochemistry result of this a proton gradient is generated across inner mitochondrial membrane. The proton gradient in turn leads to potential difference across inner mitochondrial membrane. The electrochemical gradient (low pH and positive charge on outside and high pH and negative charge inside) thus generated drives the mechanism responsible for ATP synthesis. The protons that are ejected by electron transport flows back into matrix of mitochondria through the F0 subunit of ATP synthase driven by proton gradient. The free energy released as protons flows back into matrix through the F0 subunit is used by F1 subunit for ATP synthesis from ADP and Pi (Figure 11.12). This model is supported by many experimental evidences. Fig. 11.12 A chemiosmotic model When a pair of electrons flow from NAD to O2 in the respiratory chain nearly 10 to 12 protons are pumped out. But only 6 to 8 protons are pumped out when electrons flow from FAD to O2. Since there are three complexes in the respiratory chain, each complex may extrude 3 to 4 protons when electrons flow from NAD to O2. This proton extrusion at each complex generates a pH gradient of about 0.05 units across inner mitochondrial membrane. This pH gradient causes development of 0.15 volts potential difference at each complex (Figure 11.12). As discussed earlier, this much potential difference is sufficient for ATP synthase. Thus, flow of a pair of electrons through a complex of respiratory chain is accom- panied by one molecule of ATP formation. Since respiratory chain consist of complex I, complex III, and complex IV (from NAD to O2), three ATPs are formed when electrons flow from NAD to O2. Mechanism of ATP synthase catalyzed ATP synthesis Earlier I mentioned in chemiosmotic hypothesis that energy released during proton translocation is used for ATP synthesis. To understand mechanism of ion translocation coupled ATP synthesis in molecular terms knowledge of ATP synthase structure is needed. E. Coli ATP synthase is large enzyme complex with molecular weight of 5,20,000 Kda. Its membrane extrinsic F1 portion is composed of several subunits. It contains three α, three β and one γ, δ and ε sub units. It is designated as α3 β3 γ1 δ1 ε1. Membrane embedded F0 portion is composed of one a, two b, and twelve c subunits. Two slender stalks link F1 to F0. The central stalk is formed by γ subunit and part of ε subunit. The ion channel is formed by twelve c and a subunit of F0. β subunits of F1 portion contains catalytic and binding sites (Fig. 11.13). Biological Oxidation and Respiratory Chain 281 M em α c ε γ β α b δ a Br a ne Fig. 11.13 F1F0-ATP synthase of E.Coli. ATP synthase is a mechano electrochemical enzyme. Ion translocation generates torsion in the F1 ATP synthase. Torsion in the γ subunit is generated by rotation of the c-rotor of F0 portion. Binding of protons to c subunits disturbs electrostatic equilibrium at a and c interface causing c-rotor to rotate. Due to interaction with β sub units rotation of γ sub unit is constrained. The energy from discharge of proton gradient through F0 is accumulated as torsional energy in γ sub unit of F1 portion. Thus the energy of proton gradient is stored as torsional energy in the γ sub unit. The torsional energy stored is released upon reaching threshold strain i.e. after four ion translocations. The released torsional energy causes conformational change in β sub units and thereby causing binding of ADP and P1, which leads to ATP synthesis. ATP release occurs when β sub units returns to native conforma- tion upon interaction with ε sub unit. Since ATP synthesis is related to torsional energy this mechanism is known as torsional mechanism. Unlike binding change, mechanism torsional mechanism involves irreversible mode of ATP synthesis. ATP synthase enzyme is referred as molecular machine due to rotation of sub unit γ by c-rotor in response to ion translocation. Respirasome Recent research indicates that complexes of respiratory chain are not randomly distributed in inner mitochondrial membrane. They assemble in to supra molecular structures. Complex-I, III and IV assemble into super complexes and forms network of super complexes known as respirasome. Two large super complexes and one small super complexe constitutes respirasome (Fig. 11.14). Each large super complex consists of complex-I, dimeric complex-III and two complex-IV dimmers. Hence, it is designated as I1. III2, IV4. Smaller super complex is made up of two complex-IV dimmers and one complex-III dimmer. It is designated as III2 and IV4. Further dimmers of ATP synthase also exist in mitochondria. Functional importance of super complexes Some of the advantages of super complexes over independent complexes are (a) Substrate channeling (b) Catalytic enhancement 282 Medical Biochemistry (c) Sequestration of reactive intermediates (d) Rapid intramolecular reactions Binding change mechanism of ATP synthesis In 1973, Paul D. Boyer proposed binding change mechanism to explain how the proton current and ATP synthesis are coupled. 1. In the binding change, mechanism energy stored as ion gradient across membrane containing F0 domain is used for free rotation of the c-rotor, the γ shaft and ε subunit attached to c-rotor. 2. This free rotation gets translated into binding changes in catalytic sides in the β subunit of F1 domain causing ADP and Pi to combine spontaneously to form ATP. 3. This is followed by endergonic release of ATP. 4. The novel element in Boyer’s mechanism is that the energy of proton gradient is not used in the synthesis step but only to release ATP from ATP synthase. Production and utilization of superoxide and H2O2 Production of superoxide and H2O2 They are produced as by products during reduction of O2 to water. The reduction of O2 to H2O is a multi-step process. Initially oxygen reacts with one electron, superoxide is pro- duced. If oxygen reacts with two electrons hydrogen peroxide is formed. When oxygen reacts with four electrons water is formed (Figure 11.13a). Superoxide may also formed from cytochrome P450 dependent reactions. Similarly, hydrogen peroxide may also formed from oxidases as mentioned earlier. The superoxide and hydrogen peroxide are toxic to cells. As such superoxide may not be harmful to cell but it generates free radicals like OH, OR etc., which are extremely toxic to cells. Superoxide, OH, OR, H2O2 are collectively called as reactive oxygen species (ROS). Fate of superoxide and H2O2 For the survival of cells superoxide and H2O2 must be destroyed. Superoxide is eliminated by superoxide dismutase an enzyme present in the cytosol of erythrocytes, liver, brain etc. This enzyme contains two metal ions Cu and Zn. Hydrogen peroxide is destroyed by catalase and peroxidase (Figure 11.13b). Role of super oxide and H2O2 in phagocytosis In some cells, superoxide and H2O2 are produced as a part of their normal function. For example, macrophages contribute to defense against infectious agents by phagocytosis. These cells engulf bacteria when enters into body. This engulf is followed by respiratory burst, i.e., a rapid increase in oxygen uptake. Under such conditions, O2 consumption may increase to about 50 folds. Much of this oxygen is used to generate super oxide and hydrogen peroxide. NADPH serves as donor of protons required for super oxide formation. Glucose utilization by HMP shunt, which generates NADPH also increases many folds during phagocytosis. Macrophages contain NADPH oxidase. This enzyme produces superoxide by using NADPH as source of electrons and protons. This superoxide is further reduced to H2O2 by superoxide dismutase. The superoxide and H2O2 thus produced in turn generate hypochlorite (OCl–) and hydroxyl ( OH) radicals to kill bacteria (Figure 11.13c). Biological Oxidation and Respiratory Chain 283 e– O2 4 e – , 4H + O xyg en – O2 2 e– , 2 H 2O (a ) S upe roxide 2H+ + W ate r e–, 2H + –, 2H 2e H2O2 H ydro ge n p ero xide S upe roxide dismu tase – – O2 + O2 O2 + H2O2 S upe roxides 2H+ C atalase H 2O 2 (b ) 2H2O + O 2 + + N AD P H + H N AD P – 2O2 2 O2 O xyg en N AD P H-O xida se S upe roxide S upe roxide C l – (C hloride ) – d ism u ta se 2O2 H2O2 + O2 O Cl – + H 2 O H ydro ge n H ypo chlo rite p ero xide H+ – – O2 + OCl O H + O 2 + C l– (c) S upe r H ypo H ydro xyl radical o xide chlorite O Cl – , O H B acteria D egra de d b acte ria Fig. 11.13 (a) Generation of superoxide and hydrogen peroxide. (b) Elimination of superoxide and hydrogen peroxide. (c) Molecular events of phagocytosis Fig. 11.14 A model respirasome REFERENCES 1. Morowitz, H.J. Foundation of Bioenergetics. Academic Press, New York, 1978. 2. Boyer, P.D. (Ed.). The Enzymes Vol. 13. 3rd ed. Academic Press, New York, 1976. 284 Medical Biochemistry 3. Boyer, P. Chance, B. Ernster, L. Mitchell, P. Racker, E. and Slater, E.C. Oxidative phosphorylation. Ann. Rev. Biochem. 46, 966; 1977. 4. Guengericn, F.P and Macdonald, T.L. Mechanism of Cytochrome P450 Catalysis. FASEB. J. 4, 2453-2459, 1990. 5. Hatefi, Y. The Mitochondrial Electron Transport and Oxidative Phosphorylation. Ann. Rev. Biochem. 54, 1015, 1985. 6. Slater, E.C. The Mechanism of Conservation of Energy of Biological Oxidations. Eur. J. Biochem. 166, 489, 1987. 7. Boyer, P.D. The Unusual Enzymology of ATP Synthase. Biochemistry. 26, 8503, 1987. 8. Chanock, S.F.J. et al. The Respiratory Burst Oxidase. J. Biol. Chem. 269, 24519, 1994. 9. Yankouskaya, V. et al. Architecture of Succinate Dehydrogenase and Reactive Oxygen Species Generation. Science 299, 700-704, 2003. 10. Decoursey, T.E. et al. The Voltage Dependence of NADPH Oxidase Reveals Phagocytes Need Proton Channels. Nature 422, 531-534, 2003. 11. Ruitenberg, M. et al. Reduction of Cytochrome c Oxidase by Second Electron leads to proton translocation. Nature 417, 99-102, 2002. 12. Eberhardt, Manfred. K. Reactive Oxygen Metabolites. CRC Press, 2000. 13. Stadtman, E.R. and Chock. P. Boon, Eds. Current Topics in Cellular regulation. Aca- demic Press, 2000. 14. Reikokagawa et al. The Structure of Bovine F1-ATPase Inhibited by ADP and Beryllium fluoride. The EMBO Journal. 23, 2734-2744, 2004. 15. Aronold, I. et al. Yeast Mitochondrial F1, F0-ATP Synthase Exist as dimer. Identification of three dimmer specific subunits. The EMBO Journal. 17, 7170-7178, 1998. 16. Diez, M. et al. Proton Powered Subunit Rotation in Single Membrane Bound F1, F0-ATP synthase. Nature Struct. Mol. Biol. 11, 135-141, 2004. EXERCISES ESSAY QUESTIONS 1. Describe various enzymes, coenzymes and carrier molecules involved in biological oxidation- reduction reactions. 2. Describe respiratory chain. 3. Write an essay on models of mechanism of oxidative phosphorylation. 4. Define free energy, standard free energy, exergonic and endergonic reactions and high energy compounds. Explain each one and give examples for high energy compounds. Mention importance of high energy compounds. SHORT QUESTIONS 1. Write a note on cyt P450 hydroxylase system. 2. Define high energy compounds. Explain with examples. 3. Define redox potential. Write its significance. 4. Write components of electron transport chain in the order to electron transfer. Indicate sites of phosphorylation. Biological Oxidation and Respiratory Chain 285 5. Define oxidative phosphorylation. Write principle of chemiosmotic hypothesis. 6. How super oxide is formed and utilized in the body? 7. Write enzymes involved in production and utilization of H2O2. 8. What is the role H2O2 in phagocytosis? 9. Write a note on inhibitors of oxidative phosphorylation. 10. Define reaction oxygen species (ROS). Give examples. 11. Write a note on super complexes of respiratory chain. 12. Define un couplers. Give examples. 13. Write differences between oxidative phosphorylation and substrate level phosphorylation. Give examples. 14. Write equation relating free energy and electron transfer. Write its importance. MULTIPLE CHOICE QUESTIONS 1. An example for NADP+ dependent dehydrogenase is (a) Phosphogluconate dehydrogenase. (b) Succinate dehydrogenase. (c) Acyl-CoA dehydrogenase. (d) None of these. 2. All of the following statements are correct for oxidases. Except (a) They catalyze removal of hydrogen from substrates. (b) They use oxygen as hydrogen acceptor. (c) They produce H2O2. (d) They produce H2O. 3. In iron-sulfur proteins (a) Iron is complexed with organic sulfur. (b) Iron is complexed with inorganic sulfur. (c) Iron is complexed with organic and inorganic sulfur. (d) Iron is complexed with proteins. 4. Which of the following is correct for endergonic reaction. (a) It occurs with release of energy. (b) Its ∆G is negative. (c) It occurs when energy supplied. (d) It occurs with decrease in free energy. 5. Phagocytosis involves (a) Production of superoxide. (b) Production of H2O2. (c) Production of superoxide and H2O2. (d) None of these. FILL IN THE BLANKS 1. Excess O2 is toxic to cells. So it is used in.............. treatment. 2. ATP is called as energy................ of the cell. 3. High redox potential indicates tendency of redox pair to.................... electrons. 4. P:O ratio is............... when a substance is oxidized by NAD+ dependent dehydrogenase. 5. An uncoupler................ oxidation in respiratory chain from.................. 286 Medical Biochemistry 12 CHAPTER PROTEIN AND AMINO ACID METABOLISM MEDICAL AND BIOLOGICAL IMPORTANCE 1. A 70 kg human adult body contains about 12 kg of protein. 2. Body proteins have life times. They undergo degradation and re-synthesis. About 400 gm of body protein is synthesized and degraded per day i.e., about 6 gm of protein is synthesized and broken down per kg body weight per day. 3. Aged proteins, damaged or modified proteins and non-functional proteins of the body undergo degradation. Further degradation is one way of controlling enzyme activity. Hence, continuous re-synthesis and degradation of proteins is a quality control mechanism. 4. Protein degradation may play important role in shaping tissues and organs during pregnancy and development. 5. In starvation, diabetes and tissue injury, protein degradation is more. 6. Protein synthesis and degradation is an integral part of cellular adaptation to changed environment. 7. Plasma free amino acid concentration ranges from 40 to 60 mg%. Excess amino acids can not be stored in the body. First amino group is extracted as ammonia and then carbon skeleton is oxidized to produce energy. In starvation carbon skeletons are used for glucose formation. Carbon skeletons of some amino acids produce acetyl-CoA as end product. 8. Ammonia, which is toxic to cells is converted to urea in the liver. Conversion of ammonia to urea is impaired in some inherited diseases and liver disease. 9. Amino acids are needed for the formation of specialized products like hormones, purines, pyrimidines, porphyrins, vitamins, amines, creatine and glutathione. 10. Amino acid degradation is impaired in several inherited diseases due to lack of enzymes. 11. Amino acid degradation is more in starvation, diabetes and high protein diet. 12. Some cancer cells have high amino acid (aspargine) requirement. Protein turn over In all forms of life, proteins once formed may not remain forever. Like intermediates of metabolic path ways, proteins are synthesized and degraded. Hence, body protein is in 286 Protein and Amino acid Metabolism 287 dynamic state. Continuous synthesis and degradation of protein is called as protein turnover. The rates of protein synthesis and degradation vary according to physiological needs. The rate of protein synthesis is high during growth, lactation and post operative recovery. In starvation, cancer, fever and during morphogenesis rate of degradation of protein is more. Eventhough, protein turnover involves synthesis as well as degradation of protein, protein degradation, amino acid degradation and formation of non-essential amino acids are detailed in this chapter. Protein synthesis is detailed in chapter-18. Of course conversion of aminoacids to special products is also included in this chapter. Protein half life Body proteins have life times. Life time of a protein is expressed in terms of half-life. It is defined as time required for initial amount of protein to be reduced to half. Half life of proteins ranges from minutes to years. For example, lens crystalline have very long half life whereas regulatory enzymes have very short half life. Half life of proteins may be increased or decreased depending on call needs. Half lifes (T 1/2) of some proteins are given below. Type of protein Half life (T½ ) Muscle proteins 160 days Body proteins 80 days Serum proteins 10 days Liver proteins 6 days LDH, cytochromes >100 hours HMG-CoA reductase ' r~.' --- " ~y _............-... j-"........ _- "" r~.' Too. ' - ~ -y - ~ --j. ~. , ~ - Fig. 20.9 Polymerase chain reaction (3 cycles) 3. PCR is used to detect infectious agents in the body because infenctions are due to presence of (viral or bacterial) foreign DNA. 4. PCR is used for prenatal diagnosis of genetic diseases, which are due to alterations in DNA like sickle cell anemia, hemophilia etc. 5. PCR is used to detect certain cancers like leukemia, thyroid cancer etc. 6. PCR is used for tissue typing which is essential for organ transplantation. 7. In the forensic work amplification of little DNA recovered from the suspect or from the crime site by PCR allows generation of sufficient DNA for finger printing. 8. DNA recovered from archaelogical materials (sites) is amplified by PCR and used to study evolution or civilization. 9. PCR can be used to create extinct animals like dinosaurs by amplifing DNA recovered from fossile materials. 10. Reverse transcriptase polymerase chain reaction (RT-PCR) is used to amplify RNA. 486 Medical Biochemistry 11. PCR and DNA polymorphism Now several PCR based assays are developed to detect DNA variations or DNA polymorphism. Some examples are (a) Randomly amplified polymorphic DNA (RAPD) (b) Amplified fragment length polymorphism (AFLP) (c) Sequence related amplified polymorphism (SRAP) etc. RAPD The generation of RAPDs involves use of single short random oligoneucliotides. When these random primers are mixed with sample DNA and subjected to PCR amplification of several fragments occurs. The DNA amplification with random primers expose polymorphisms distributed through out the genome. RAPD is also used in genome mapping and gene tagging. AFLP This PCR-based technique permits inspection of polymorphism at large number of loci with in short period of time and requires very small amount of DNA. AFLP is potentially used in genome finger printing and mapping. Restriction fragment length polymorphism (RFLP) It is another technique based on hybridization principle. DNA is a polymorphic molecule, i.e., exist in several forms. DNA of an individual varies from others. Sequence of DNA of an individual is unique. Further, mutations in DNA generates polymorphic DNA in same individual, which occurs in diseases. So, DNA polymorphism is due to variations in sequence. When DNA of an individual is subjected to digestion with restriction enzyme fragments of varying sizes or lengths that are unique to inviduals sequence or cell are produced. RFLP may also result from presence of variable numbers of tandem repeates (VNTR) in DNA. These are short sequences of DNA that are scattered locations in genome and repeated in tandem. The number of these repeats are unique to individual. When DNA of two individuals is subjected to digestion with restriction enzymes fragments that vary in length and number are generated. Therefore, RFLP of two individuals results from the differences in the location and number of cleavage sites. Differences in DNA of two individual may be due to evolutionary changes. RFLP is similar to southern blotting in many aspects. Initial step of RFLP involves digestion of more DNA samples with restriction enzymes where as in southern blotting only one DNA sample is digested. Rest of the steps of RFLP are those of southern blotting. Hence, in RFLP next step probes are used for hybridization. Probes hybridizes with fragment containing complementary sequences. Then polymorphisms are detected by presence or absence of bands after hybridization. Applications 1. RFLP is used as a diagnostic test of inherited disease. For example, HbS: In HbS gene there is loss of one restriction site for restriction enzyme due to mutation where as normal HbA gene has two cleavage sites. So, RFLP of sickle-cell anemia patient shows two bands where as in RFLP of normal individual three bands appear. 2. RFLP is also used to identify chromosomal difference. Recombinant DNA Technology 487 3. RFLP is used for isolation and sequencing of closely related genes. 4. RFLP is combination with PCR is used to detect DNA variations. Bioinformatics 1. It is the combination of IT (Information Technology) and Life Sciences like Biochemistry, Molecular Biology, Biotechnology etc. 2. It is defined as application of information technology and science for organisation management, mining and use of life sciences. 3. Main application areas of bioinformatics are genomics, proteomics, pharmacogenomics, chemiinformatics etc. 4. One of the earliest application are a of bioinformatics is in drug design process. Bioinformatics revolutionized traditional approach of drug discovery from target discovery and screening to discovery and development of therapeutic agents whose role in prevention of cure of a disease is well validated. Further, drugs so designed have less failures. 5. Following steps of bioinformatics based method of designing drug that is an enzyme inhibitor. (a) Selection of chemical fragments from molecular library. (b) Assembly of chemical fragments in a piece-wise manner into possible inhibitor molecule. (c) Using docking algorithm all the possible inhibitor molecules are screened to select highly potent inhibitor which precisely fits in the binding cavity of enzyme. 6. Knowledge of genome sequence allows structure activity based drug designing. Following are steps of drug designing process, which involves genome sequence knowledge. (a) Determination of protein sequence using DNA sequence. (b) Prediction algorithms are used to visualise structure adopted by the protein molecule. (c) Using docking algorithm a molecule that binds and alters protein function is identified as a drug. REFERENCES 1. Wu, K., Grossman, L. and Moldave, K. (Eds.). Recombinant DNA methodology. Academic Press, New York, 1989. 2. Berger, S.L. and Kimmal, A.R. Methods in Enzymology, Vol. 152, Academic Press, California, 1987. 3. Kantoff, P.W. Prospects for gene therapy for immuno deficiency disease. Ann. Rev. Immunol, 6, 58–94, 1988. 4. Agarwal, S. and Jang, J. GEM 91. An anti-sense oligo nucleotide phosphorothioate as a therapeutic agent for AIDS. Anti-sense Res. Dev. 2, 261–266, 1992. 5. Rangarajan, P.N. and Padmanabhan, G. Gene therapy: principles, practice, problems and prospects. Curr. Sci. 71 (5), 360–367, 1996. 6. Marx, J.L. DNA finger printing takes witness stand. Science 240, 1616–1618, 1988. 488 Medical Biochemistry 7. Mullus, K.B. The unusual origin of polymerase chain reaction. Sci. Am. P. 56, April 1990. 8. Meada, M.H. Dairy Gene. The Sciences. New York Academy of Sciences, New York, P. 21, October 1997. 9. Michel, K. and Schmidtke. J. DNA finger printing. BIOS Scientific Pub. USA, Canada, 1994. 10. Schena, Mark, (Ed.) DNA micro arrays: A practical appraoch, Oxford University Press, 1999. 11. Siebert, P. (Ed.). The PCR technique. RT-PCR. Eaton Publishing, MA, USA, 1998. 12. Mcpherson, M-J and Moller, S.G. PCR, Springer-Verlag Talos, 2000. 13. Dieffenbach, Carl. W. and Gabriel, S.D. PCR primer: A Laboratory manual. Cold Spring Harbor Laboratory Press, NY, 2003. 14. K. Shah et al. Molecular imaging of gene therapy for cancer. Gene Therapy. 11, 1175– 1187, 2004. 15. Li, G. and Quiors. Sequence related amplified polymorphism (SRAP) a new marker system based on simple PCR reaction: its application to mapping and gene tagging in Brassica. Theor. Appl. Genet. 103, 455–461, 2001. 16. vander wurff. A.W.G. Chan, Y.L. vanstraalan, N.M. and Schouten. J. TE-AFLP; Combining rapidity and robustness in DNA finger printing. Nucleic acids Research. 28, 105–109, 2000. 17. Song, H. et al. Neural stem cells from adult hippo campus develop essential properties of functional CNS neurons. Nature 417, 39–44, 2002. 18. Gage, F.H. Mammalian neural stem cells. Science. 287, 1433–1438, 2000. 19. Lesk, A.M. (Ed.). Introduction to Bioinformatics. Oxford University Press, New York, 2002. 20. Isner, J.M. Myocardial gene therapy. Nature. 415, 234–239, 2002. 21. Austin, C.P. et al. The knockout mouse project. Nature Genetics. 36, 921–924, 2004. 22. Kubota, C. et al. Serial bull cloning by somatic cell nuclear transfer. Nature Biotechnology. 22, 693–694, 2004. 23. Susan M. Rhind et al. Human cloning: can it be made safe. Nat. Rev. Geneti. 4, 855–864, 2003. 24. Rebeca J. Morris et al. Capturing and profiling adult hair follicle stem cells. Nature Biotechnology. 22, 411–417, 2004. 25. Arekawa, T. et al. Efficacy of food plant based oral cholera toxin B sub unit vaccine. Nature. Biotechnol. 16, 292–297, 1998. 26. Karatzas, C.N. Designer milk from transgenic clones. Nature. Biotechnol. 21, 138–139, 2003. 27. Nishimura, E. K. et al. Mechanism of hair graying: Incomplete melanocyte stem cell maintenance in the Niche. Science. 307, 720-724, 2005. EXERCISES ESSAY QUESTIONS 1. Define recombinant DNA. Explain steps of recombinant DNA technology used for production of human gene products in a biotechnology company. Recombinant DNA Technology 489 2. Give an account of gene therapy. 3. Describe production and application of hybridomas. 4. Describe hybridization techniques. 5. Describe polymerase chain reaction (PCR). Short questions 1. Write steps involved in production of transgenic animals. 2. Define cloning. Write steps necessary for production of cloned sheep. 3. Write a note on DNA vaccines. 4. Define edible vaccines. Write steps of edible vaccination. 5. Write a biosensor working principle. Name components of a biosensor and write their applications. 6. Explain western blot technique. Write its significance. 7. Write recombinant DNA technology applications. Fill in the blanks 1. Recombinant DNA technology may lead to creation of................. species. 32 2. cDNA probe is................. molecule prepared from P labelled nucleoside triphosphates. 3. Cloning is a................. reproduction of mammals. 4.................. cells are immortal. 5. ACAAACT is repetitive sequence of fruit fly.................. 490 Medical Biochemistry 21 CHAPTER CANCER AND AIDS MEDICAL AND BIOLOGICAL IMPORTANCE 1. Cancer is a major health problem affecting humans throughout the world. Several types of cancers affecting major organs like lung, brain, kidney, colon, breast, oesophagus and stomach have been identified. 2. Rate of incidence of cancer of particular organ in particular population depends on several factors like age, sex, dietary habits, environment, geographical location, genetic make up, culture, physical exercise etc. For example in India oral cancer is common in betal nut chewing regions and in reverse smokers. Stomach cancer is more in Japanese and Chinese people. Colon cancer is common in advanced countries and lung cancer is common in smokers. Old people are more prone to any type of cancer. Brain cancer and blood cancer are common in children. Men above 50 are prone to prostate cancer. Women above 45 are prone to breast, ovarian and cervical cancers. 3. Rate of incidence of cancer of particular organ varies from developed countries to developing countries. (Table 21.1). Lung and colorectal cancers are high in developed countries while stomach and cervical cancer are more in developing countries. Further in India pharyngeal cancers are high in Western India where as stomach cancers are more common in Southern India. Nearly 10 million new cases of cancer are diagnosed globally every year. It is estimated that by 2020 ten million persons would die of cancer every year World wide. Table 21.1 Cancer incidence rate in developed and developing Countries Cancer site Developed countries Developing countries Lung 62 24 Colon and rectum 20-45 2-8 Stomach 10 60 Cervix 14 30 Prostate 30 10 Mouth and pharynx 13 25 490 Cancer and AIDS 491 4. Nuclear architecture is altered in cancer cells. New anticancer drugs that revert these changes may be developed. Like wise new tumour marker based on nuclear structural changes may be used in cancer diagnosis. 5. Extensive research carried out for the last two decades on various types of cancer led to development of proper treatment for at least some types of cancers. However, it greatly expanded our knowledge on molecular mechanism of cancer. 6. AIDS is another major health problem that surfaced around second half 20th century. According to WHO (World Health Organization) estimation about 20 million people are affected by AIDS. At least about 5-10000 people get infected for every 24 hours. Spread of this infectious disease also depends on several factors. In developing countries it is spreading faster due to prevalent socioeconomic conditions. 7. Though the various facets of cancer and AIDS are being probed thoroughly for the last two decades proper cure is not in sight particularly for AIDS. CANCER Growth of all types of cells is controlled in the body. If the growth of cell is not controlled they continue to proliferate which leads to malignancy. So cancer is malignant growth (un controlled growth) of cells. Malignant growth of cell is also called as tumour. Cancer of a particular organ or tissue develops when the cells of that organ have lost growth control. In addition cancer cells has other abilities a) Invasion b) Metastasis. Cancer cells are carried to other parts of the body by circulation where they develop further. So, Cancer of one organ if not detected can spread to other organs (Figure 21.1). Fig. 21.1 Cancer development from normal cell. Nomenclature and classification of Cancers Generally cancers are named according to the organ affected. However they are classified based on the three embryonic germ layers from which tissue or organ is derived. 1. Carcinomas Are the cancer of cells or organs derived from either ectoderm or endoderm. Cancers of epithelial tissues, nervous tissues, glands etc. are named as carcinomas. 492 Medical Biochemistry Example: (a) Adenocarcinoma: Cancer of gland. (b) Squamous cell carcinoma: Cancer of squamous cells of epithelial tissues. (c) Gliomas: Cancer of brain nervous tissues. 2. Sarcomas Are cancers of tissues of mesodermal origin. Generally cancers of bone, cartilage, connective tissue, muscle etc are called as sarcomas. Examples: (a) Osteosarcoma: Bone cancer. (b) Fibrosarcoma: Connective tissue cancer. Cancer is primarily due to DNA damage or damage of genes. DNA damage may result from the action of biological, chemical, physical and environmental agents on DNA. Incidence of cancer also depends on the genetic make up of an individual. Cancer genes Oncogenes. Are genes responsible for development of cancer. Proto oncogenes. They are precursors of oncogenes. They are converted to oncogenes by activation. Tumour suppressor genes. They are present in normal healthy people. Products of them prevent cancer development. The product of oncogenes disturbs the normal cell growth control mechanism leading to cancer. Usually products of oncogenes are protein kinases that phosphorylate tyrosine residues of proteins. (Tyrosine kinase.) Both cellular and viral oncogenes are found. Examples for oncogenes and protooncogenes are given below. 1. Cellular oncogene that causes rat sarcoma is designated as c-ras oncogene. Likewise c-ras protooncogene. 2. Viral oncogene that causes rat sarcoma is designated as v-ras oncogene. Likewise v-ras protooncogene. 3. Oncogene of rouse sarcoma is designated as src-oncogene. 4. Oncogene of simian sarcoma is designated sis-oncogene. 5. Oncogene of chicken myelocytoma is designated myc-oncogene. Carcinogenesis By several ways carcinogenesis occurs in humans and other animals. Usually they are named according causative agent or factor. Different types of carcinogenesis are given below: 1. Biological agents that cause cancer or biological (viral) carcinogenesis. Some DNA and RNA viruses are carcinogenic and hence they are called as oncogenic viruses. When normal cells are cultured with oncogenic viruses, the normal cells are trans- formed into cancer (tumour) cells. Oncogenes of the viruses are responsible for the development of cancer. Cancer and AIDS 493 Examples 1. Hepatitis B virus cause liver cancer in humans. 2. Retro viruses also cause cancer in humans. 2. Chemical carcinogens or mutagens or chemical carcinogenesis. Many chemical substances cause mutations in DNA. They are called mutagens. Sometimes this muta- tion in DNA may convert normal cell to cancer cell. Then they are called as carcino- gens. Examples 1. Cigarette smoke causes lung cancer in humans. 2. Aflatoxins are carcinogens. 3. Nitrosamine, Benzapyrins and asbestos also cause cancer. 3. Physical agents that cause cancer or physical carcinogenesis. Exposure to radia- tion may damage DNA. UV light exposure causes mutation in DNA of skin cells. Mutant DNA mediates carcinogenesis by activation of oncogenes which leads to development of cancer of skin or multiple tumours of skin. Cancer due to genetic factors: Some genes in DNA are associated with development of cancer in susceptible individu- als. Examples: 1. Retinoblastoma, cancer of eye develops in people carrying RBI gene. 2. Wilm’s tumour, kidney cancer develops in children having gene WTI. Activation of protooncogene to oncogene By several ways activation of protooncogene to oncogene can occur. Some of them are given below. (a) Point mutation. Point mutation converts protooncogene to oncogene. Human bladder carcinoma is due to point mutation. Mutation may be due to error during replication. (b) Gene amplification. Amplification of oncogenes results in the formation of products of these genes by several folds. This in turn converts normal cells to cancer cells. (c) Chromosomal translocation, promoter/enhancer insertion also leads to activation of protooncogene to oncogene. Mechanism of action of oncogenes or Mechanism of carcinogenesis The product of oncogene converts normal cell to cancer cell by several ways. 1. The product of c-ras oncogene is a less active GTPase. This leads to prolonged activation of adenylate cyclase and hence activities of cAMP dependent proteinkinase. As a result cellular metabolism is altered and normal cell is transformed into cancer cell. 2. Myc-oncogene product is DNA binding protein or transcription factor (TF). It regulates expression of cell cycle genes. As a result cell cycle is altered. 3. Src-oncogene product is tyrosinekinase. It phosphorylates cyclins and cyclin dependent kinases of cell cycle. This results in cell cycle alteration. 4. Some oncogene products are polypeptide growth factors that affect cell cycle and mitosis. 494 Medical Biochemistry Mechanism of virus mediated carcinogenesis 1. Several human tumour viruses induce immortalization of human tissue cells. It is followed by malignant conversion which involves several steps. 2. Human papilloma viruses (HPV), human T-lymphocyte virus (HTLV) possess defined oncogenes that stimulate proliferation of human cells. 3. Human papilloma virus causes cervical cancer. Cancer of cervix is the number one cancer in Indian women. In India about 100,000 women develop this cancer every year. 4. Human T-lymphocyte virus causes T-lymphocyte leukemia. 5. The oncogenes of HPV are E6 and E7. They are able to immortalize keratinocytes. They contain all necessary information for immortalization. 6. The E7 protein releases transcription factor which activates genes engaged in cell cycle progression. 7. The E6 protein binds P53 and abolishes its tumour suppressive and Trans activational properties. It also promotes ubiquitinisation of P53 and its subsequent proteolysis. 8. Thus E6 and E7 are able to immortalize cells independently and both genes cooperate effectively in immortalization of cells. Metabolism of carcinogen Metabolism of carcinogen after entering the body is mainly directed towards producing metabolites which can be excreted. The enzyme systems of phase-I and phase-II reactions of metabolism of xenobiotics are mainly responsible for the formation of excretory metabolites. Sometimes these compounds lead to tumour formation. If it leads to formation of malignant tumour then it is known as Cancer. Cytochrome P450 enzymes of phase-I are involved in the formation of a carcinogen. Mechanism of a carcinogen mediated carcinogenesis 1. Carcinogenesis by a carcinogen involves several steps. 2. First step is the induction of molecular lesion. 3. Second step is the fixation of molecular lesion by DNA replication. 4. The ultimate carcinogenic forms of carcinogens are highly reactive electrophiles which are reactive towards DNA. 5. They bind covalently with DNA to produce DNA adduct. 6. This type of DNA modification is major driving force for cancer development. Nuclear structure of Cancer cells 1. In cancer cells nuclear architecture is altered. These alterations are characteristics of tumour type. 2. Components of nuclear matrix play a role in organization of chromosomes and nuclear components. Protein composition of nuclear matrix is altered in cancer cells. 3. Oncogenes induce tumor-specific nuclear changes and these in turn changes gene regu- lation. 4. In cancer cells chromosomal territories and gene loci are changed. Cancer and AIDS 495 5. Structural changes in tumour cells lead to changes in nucleoli and perinuclear compart- ment. 6. These changes can be used as potential tumour markers and targets for anti-cancer drugs. Treatment of cancer Several types of treatments are available for cancer management. Some are given below. Cancer gene therapy is explained in earlier chapter. 1. Chemotherapy Compounds that block replication of cells and anti metabolites that block nucleotide biosynthesis are used as anticancer agents or in chemotherapy of cancer. (a) Mercapto purine. It is a purine analog used in the treatment of luekaemia. It is converted into nucleotide in vivo and incorporated into nucleic acids and interferes with replication. (b) Fluoro uracil. It is a pyrimidine analog and used in the treatment of colorectal cancer. In vivo it is converted fluorodeoxy uridine phosphate and inhibits replication. (c) Methotrexate. It is a folic acid analog and used in the treatment of chorio carcinoma. (d) Azaserine. It is a glutamine analog used in cancer treatment. It blocks nucleic acid biosynthesis (replication) by inhibiting glutamine dependent metabolic reactions. (e) Acivicin. Another glutamine analog used as anticancer agent. It is a competitive inhibi- tor of glutamine utilizing enzyme. Methotrexate, azaserine and acivicin are anti metabolites used in cancer treatment. They are called as anti metabolites because they block nucleic acid synthesis by anatgonizing metabolic role of glutamine. 2. Radiotherapy Radiation can break phosphodiester linkages of DNA and interferes with replication process. As a result growth of cancer cells can come down. Based on this principle radiation is used to treat tumours. 3. Surgery It is the treatment of choice in the advanced stages of cancer. Cancer (tumour) tissue is removed by surgery. Usually surgery is performed with operataing microscope. 4. Photo chemotherapy It is a newly introduced treatment for cancer. It uses a photosensitive drug and laser light to destroy cancer cells. 5. Suicide Gene Therapy (Molecular surgery) It is a kind of gene therapy used in the treatment of solid tumours where therapeutic gene is targeted at tumour cells killing cells which expressing it. It is also known as molecular surgery. The suicide genes are enzymes which activates low toxic prodrug to toxic potent drug. Herpes simplex thymidine kinase (HSTK) and cytosine deaminase (CD) are two such enzymes. HSTK converts non-toxic anti-viral drug ganciclovir to toxic form by phosphorylation. 496 Medical Biochemistry CD converts non-toxic fluorocytosine into toxic fluorouracil. Vectors carrying genes of these enzymes are injected directly into tumour. It is followed by intratumoural injection of prodrug. Tumour Markers Cancer (tumour or malignant) cells produce abnormal substances. Usually these substances are not produced by normal cells. The abnormal substances produced by the cancer cells are enzymes, hormones and proteins. These substances are released into blood by cancer cells. As a result their level in blood rises. Measurement of these substances in blood or serum provides useful information about cancer. Hence, they are called as tumour markers, Nowa- days measurement of tumour markers in blood is an integral part of oncology. Tumour marker measurement is used in (a) Detection of cancer. (b) Diagnosis of cancer. (c) Prognosis of cancer. (d) Determination of cancer stage. (e) Determination of location of cancer in the body. (f) Determination of organ involved in cancer. (g) Cancer therapy. Some clinically important tumour markers are 1. α-Feto protein(AFP). It is a plasma protein and usually absent in normal people plasma. It is tumour marker for liver cancer and germ cell cancer. 2. Calcitonin. It is a hormone. It is tumour marker for thyroid cancer. 3. Carcino embryonic antigen (CEA). It is a protein and it is tumour marker for lung cancer, breast cancer, colon cancer and pancreas cancer. 4. Human chorionic gonodotropin (HCG). It is tropic hormone. It is tumour marker for germ cell cancer and trophoblast cancer. 5. Acid phosphatase. It is tumour marker for prostate cancer. 6. High mobility group chromosomal proteins (HMGCP). They are family of non- histone chromosomal proteins that serve as architectural elements in chromatin. In normal tissues these proteins are expressed at very low levels. Their level is elevated in many human cancers. This small molecular weight proteins' expression is increased in neoplastic transformation of cells and metastatic of tumour progression. They can serve as novel diagnostic tumour markers. Disadvantages of tumour markers 1. These tumour markers usually detect cancer at advanced stage. So they are of little help in saving lives. 2. A given marker is useful in the detection of only one type of cancer. 3. Sometimes measurement of more than one type of tumour marker may be helpful or required. Cancer and AIDS 497 AIDS It is the abbreviated form of Acquired Immuno Deficiency Syndrome. It is an acquired disease. It is an infectious disease. In this disease body immune or defense system weakens. It is named as syndrome because full blown disease makes up many diseases. AIDS is caused by a virus called as Human Immuno Deficiency Virus type-I (HIV-I). It is a retrovirus, It consists of RNA which is surrounded by two types of proteins. The RNA core is enveloped in membrane lipid bilayer containing glycoproteins (Figure 21.2). When HIV infects humans it infects T-cells of lymphocytes which form an important part of immune system. The lymphocytes (T-cells) fight diseases by killing disease causing agents. The cell surface of T-cells contains a glycoprotein receptor known as CD-4 receptor. The T- cells are also called as CD-4 cells because of this. AIDS virus attacks CD-4 cells and kills them. So when a person is infected with HIV for prolonged period his CD-4 cell count decreases and he is susceptible to infections. Fig. 21.2 Structure of AIDS virus HIV Life cycle 1. HIV genetic material is single stranded RNA. 2. When HIV enters into body, it gets attached to T-cell through CD-4 receptor. 3. Then HIV internalizes in the cell after fusing with membrane of CD-4 cell. Its contents are released into the CD-4 cell. 4. The genetic material of HIV is transformed into DNA by reverse transcriptase. 5. The HIV DNA is integrated into host DNA. 6. Expression of HIV RNA and translation of RNA produces proproteins in the CD-4 cell. 7. Pro proteins are processed by protease (HIV) to perfect proteins of HIV. 8. Assembling of RNA and HIV proteins into new HIV particles. 9. Newly formed HIV comes out of CD-4 cell by killing it or when CD-4 cell dies. Various events involved in HIV life cycle are shown in Figure 21.3. 498 Medical Biochemistry Fig. 21.3 HIV Life cycle Symptoms of AIDS In the early phase HIV infected people develop flu associated symptoms like fever, headache, swollen lymph glands, stomach ache and swollen joints. These initial symptoms subside after few days and infected people remain normal for a period ranging from 6 months to 10 years or even longer. During this period HIV multiplies in the body and kills many T-cells. As a result CD-4 cells count decrease. In normal people the T-cell count is between 500 to 1500/ ml of blood. As the T-cell (CD-4) count decreases in the blood HIV infection symptoms like night sweats, diarrhoea and fever surfaces and remain for few days to few weeks. HIV infection becomes AIDS when T-cell count goes down to 200/ml of blood. At this stage, HIV infected people contact opportunistic infections like tuberculosis, pneumonia, weight loss, tumours and fungal infections. Recovery from these conditions is slow and requires extensive treatment. Laboratory Diagnosis When a person is infected with HIV antibodies to HIV are produced in the body like in any other infection. So the presence of HIV antibodies in the blood indicates infection. Most of the AIDS detection tests are based on identification of HIV antibodies in the blood. Enzyme linked immunosorptive assay (ELISA) and western blot technique are used to detect HIV antibodies in blood. Some AIDS detection tests are based on genetic material of HIV. AIDS Therapy Currently AIDS treatment involves use of two classes of drugs. They are (a) Inhibitors of reverse transcriptase Since reverse transcriptase is important for replication of HIV blocking reverse transcriptase action can control HIV proliferation. Some of the reverse transcriptase inhibitors used as drugs are AZT, ddI, ddc, d4T and 3TC. Cancer and AIDS 499 (b) Protease inhibitors Since HIV protease is essential for processing of proteins, blocking of this enzyme also can arrest HIV proliferation. Some of HIV protease inhibitors used as drug are indinavir, saquinavir, ritonavir and nelfinavir. HIV AND CANCER In India, HIV-1 is mostly responsible for AIDS. HIV-2 is common in West Africa and found in India also. Both HIV-1 and HIV-2 are not directly oncogenic. However, Kaposi’s sarcoma of AIDS patients is largely an attribute of HIV. Kaposi's sarcoma (KS) is a rare tumour found only in men over sixty years in certain Eastern European and Mediterranean population. However, risk of KS in HIV infected adult male homosexuals less than sixty years old is some ten thousand fold higher than that of their counterparts in general population. With AIDS, endemic KS has become most common of all tumours in Sub Saharan Africa. The epidemology of KS before and after AIDS sug- gested a transmissible agent may underlie tumour. Human herpes virus-8 (HHV-8) or KS- associated herpes virus (KSHV) is discovered as responsible for KS in humans. KSHV DNA is found in KS biopsies alone and in HIV positive patients. Both KSHV and HIV infections are independent and highly risk factors in the development of KS in AIDS patients. The risk of KS in KSHV positive patients's increases with decreasing CD-4 T lymphocytes as occurs in AIDS. It is believed that Tat protein of HIV-1 has role in KS pathogenesis. It acts synergistically with cellular growth factors. However, KS commonly occurs in KSHV pa- tients with HIV-1 than with HIV-2 infection. In HIV positive patient KSHV is also associated with lympho proliferative disease. Burkitt’s lymphoma (BL) and Non-Hodgkin lymphoma (NHL) are two lymphomas fre- quently seen in AIDS cases. NHL in AIDS occurs in brain and BL in gut. Incidence of any cancer increases in AIDS patients due to immune suppression. The immune suppression induced by HIV accelerates progression of malignancy. Liver cancer, skin cancer, testicular cancer and treatocarcinoma are more in AIDS patients. Hence, cancers associated with AIDS are probably opportunistic neoplasms like opportunistic infections. REFERENCES 1. Weinberg, R.A. A molecular basis of cancer. Sci. Am. 249(5), 126-142, 1983. 2. Boyle, P. Nutritional factors and cancer. In Human Nutrition and Dietetics. Garrow, J.S. and James, W.P.T. (Eds.) 9th ed. Churchill Livingstone, Edinburgh, 1993. 3. Lavecchia, C. Bidoli, E. and Barra, L. Types of Cigarettes and cancer of upper digestive and respiratory tract. Cancer causes control. 1, 69-74, 1990. 4. Dwyer, M.J. Biomedical aspect of HIV and AIDS. Curr. Sci. 69(10), 823-827, 1995. 5. Wlodawer, A. and Ericikson, J.W. Structure based inhibitors of HIV-1 proteases. Ann. Rev. Biochem. 62, 543-585, 1993. 6. Roberts, N.A. Drug resistance patterns of saquinavir and other HIV protease inhibitors. AIDS, 9, 527-532, 1995. 7. J. Cohen, HIV/AIDS in Asia, Science, June, 2004. 500 Medical Biochemistry 8. HIV/AIDS in India, Science, April 2004. 9. Ryan, K.M. and Vousden, K. Cancer: Pinning a change on P53. Nature 419, 795-797,2002. 10. Mikail, V. Blagosklonny. Cell immortality and Hall marks of cancer. Cell cycle. 2, 296- 299, 2003. 11. Kristi, G. Bache. et al. Defective down regulation of receptor tyrosine kinases in cancer. The EMBO Journal. 23, 2707-2714, 2004. 12. Donald, W.K. et al. (Eds.) 6th ed. Cancer medicine, BC Decker, 2003. 13. Veldwij, K.M.R. et al. Suicide gene therapy of Sarcoma cell lines using recombinant adeno associated virus vectors. Cancer gene therapy. 11, 577-580, 2004. 14. Bandura, J.L. and Calvi, B.R. Duplication of genome in normal and cancer cell cycle. Cancer Biol. Ther. 1, 8-13, 2002. 15. Zink, D. et al. Nuclear structure in cancer cells. Nat. Rev. Cancer. 4, 677-687, 2004. 16. Parkin, D.M. et al. Estimating world cancer burden. Int. J., Cancer. 4, 153-156, 2001. 17. Mathew, A. Cancer registration with emphasis on Indian Scenario. In ‘Basic information for cancer registry documentation’ (Ed. Mathew. A.), Regional Cancer Centre, Trivendrum, pp11-17, 2003. 18. Davis, M.I. et al. Crystal structure of prostate specific antigen a tumor marker and peptidase. Proc. Natl. Acad. Sci. USA. 102, 5881-5986, 2005. EXERCISES SHORT QUESTIONS 1. Define oncogenes, protooncogenes and tumour suppressor genes. Give examples for chemical carcinogens. Explain how they cause cancer. 2. What are tumour markers ? Give an example and its clinical importance. 3. Expand HIV. Write a note on HIV life cycle. 4. Write symptoms and treatments available for AIDS. 22 CHAPTER PORPHYRIN AND HAEMOGLOBIN METABOLISM MEDICAL AND BIOLOGICAL IMPORTANCE 1. Porphyrins are present in biological fluids like blood, bile, urine and feces of animals and invertebrates. They are also found in plants and bacteria. 2. Porphyrins are components of hemeproteins of animals and invertebrates. Heme is metalloporphyrin. It contains metal iron in the centre of porphyrin ring. Hence heme proteins are referred as metalloporphyrinoproteins. 3. Hemeproteins like hemoglobin and myoglobin are involved in O2 transport in animals and vertebrates. 4. In invertebrates erythrocruorins which are also hemeproteins are responsible for the O2 transport. 5. Hemeproteins like cytochromes and cytochrome oxidase are components of respiratory chain and involved in electron transport. 6. Cytochrome P450 which is involved in detoxification of drugs is a hemeprotein. 7. Some hemeproteins are involved in metabolism. For example tryptophan dioxygenase an enzyme of tryptophan catabolism is a metelloprophyrinoprotein and cyclooxygenase an enzyme of prostaglandin synthesis is a hemeprotein. 8. Hemeproteins like catalase and peroxidase are involved in the removal of H2O2. 9. In plants porphyrins are components of chlorophyll and phycobilins. 10. In bacteria porphyrins are components of cyanocobalamin. 11. A group of inherited diseases known as porphyrias are due to abnormalities in heme (porphyrin) biosynthesis. Lead poisoning also blocks porphyrin biosynthesis. 12. A common disease jaundice is due to excessive catabolism of porphyrins or heme con- taining compounds. Hepatitis and cancer of pancreas also can cause jaundice. 13. A group of inherited diseases known as hemoglobinopathies are due to abnormalities in production of hemoglobin. 14. Carbon monoxide a poisonous gas present in automobile exhaust works by combining with hemoglobin. 501 502 Medical Biochemistry 15. Photosensitive property of porphyrins is used in cancer photochemotherapy. 16. Bilirubin end product of heme catabolism act as antioxidant. 17. Hemoglobin is a source of protein for malarial parasite during malaria. Enzymes of hemoglobin degradation pathway are exploited for new drug design. Porphyrins Chemistry Porphyrins are derived from a parent compound porphin. Porphin is a tetrapyrrole and it is a cyclic compound. In porphin, 4 pyrroles are linked through methenyl (—CH=) bridges. Four pyrrole rings of porphin are shown with Roman numbers I, II, III and IV. Methenyl bridges are indicated by Greek numbers α, β, γ and δ. Substituent positions of I, II, III and IV rings are indicated with Indo-Arabic numbers 1, 2, 3, 4, 5, 6 and 7, 8 respectively (Fig. 22.1). The eight numbered substituent positions corresponds to eight hydrogen atoms of pyrrole rings. Short hand representation of Porphyrins Naturally occurring porphyrins contain various side chains in place of 8 hydrogen atoms. They differ only in side chains attached to four pyrrole rings. Hence Fischer proposed a short hand form for porphyrins in which only substitutions are particularly shown. In this short hand form as shown in Fig. 22.1 each pyrrole ring with numbered substituent positions is shown as bracket. The four brackets are indicated with Roman numbers and they are joined by eliminating the methenyl bridges to form cross shape. Fig. 22.1 Structures of pyrrole, porphin and porphyrin Porphyrin and Haemoglobin Metabolism 503 Porphyrins Isomers Porphyrins are intermediates of heme biosynthesis. Each porphyrin can exist in many isomeric forms which depends on kinds of side chains and arrangement of side chains. For example a porphyrin like uroporphyrin with two type of side chains acetate (A), propionate (P) can exist in four isomeric forms or four types. They are type I, type II, type III and type IV. In uroporphyrin I side chains are arranged symmetrically. In other types side chains A, P are arranged asymmetrically (Fig. 22.2). In nature I and III type porphyrins are more. However type III is predominant and more important for heme biosynthesis. Protoporphyrin with three types of side chain can exist in fifteen isomeric forms. In protoporphyrins two pyrrole rings contain methyl (M) and propionate (P) side chains. Other two pyrrole rings contain methyl (M) and Vinyl (V) side chains. Heme of hemoglobin contains protoporphyrin IX (Fig. 22.2). Fig. 22.2 (a) Short hand forms of uroporphyrin and protopor