Harper's Illustrated Biochemistry, 26th Edition PDF

fm01.qxd 3/16/04 11:10 AM Page i a LANGE medical book Harper’s Illustrated Biochemistry twenty-sixth edition Robert K. Murray, MD, PhD Professor (Emeritus) of Biochemistry University of Toron...

fm01.qxd 3/16/04 11:10 AM Page i a LANGE medical book Harper’s Illustrated Biochemistry twenty-sixth edition Robert K. Murray, MD, PhD Professor (Emeritus) of Biochemistry University of Toronto Toronto, Ontario Daryl K. Granner, MD Joe C. Davis Professor of Biomedical Science Director, Vanderbilt Diabetes Center Professor of Molecular Physiology and Biophysics and of Medicine Vanderbilt University Nashville, Tennessee Peter A. Mayes, PhD, DSc Emeritus Professor of Veterinary Biochemistry Royal Veterinary College University of London London Victor W. Rodwell, PhD Professor of Biochemistry Purdue University West Lafayette, Indiana Lange Medical Books/McGraw-Hill Medical Publishing Division New York Chicago San Francisco Lisbon London Madrid Mexico City Milan New Delhi San Juan Seoul Singapore Sydney Toronto fm01.qxd 3/16/04 11:10 AM Page ii Harper’s Illustrated Biochemistry, Twenty-Sixth Edition Copyright © 2003 by The McGraw-Hill Companies, Inc. All rights reserved. Printed in the United States of America. Except as permitted under the United States Copyright Act of 1976, no part of this publication may be reproduced or distributed in any form or by any means, or stored in a data base or retrieval system, without the prior written permission of the publisher. Previous editions copyright © 2000, 1996, 1993, 1990 by Appleton & Lange; copyright © 1988 by Lange Medical Publications. 2 3 4 5 6 7 8 9 0 DOC/DOC 0 9 8 7 6 5 4 3 ISBN 0-07-138901-6 ISSN 1043-9811 Notice Medicine is an ever-changing science. As new research and clinical experience broaden our knowledge, changes in treat- ment and drug therapy are required. The authors and the publisher of this work have checked with sources believed to be reliable in their efforts to provide information that is complete and generally in accord with the standards accepted at the time of publication. However, in view of the possibility of human error or changes in medical sciences, neither the authors nor the publisher nor any other party who has been involved in the preparation or publication of this work warrants that the information contained herein is in every respect accurate or complete, and they disclaim all responsibility for any er- rors or omissions or for the results obtained from use of the information contained in this work. Readers are encouraged to confirm the information contained herein with other sources. For example and in particular, readers are advised to check the product information sheet included in the package of each drug they plan to administer to be certain that the information contained in this work is accurate and that changes have not been made in the recommended dose or in the contraindications for administration. This recommendation is of particular importance in connection with new or infre- quently used drugs. This book was set in Garamond by Pine Tree Composition The editors were Janet Foltin, Jim Ransom, and Janene Matragrano Oransky. The production supervisor was Phil Galea. The illustration manager was Charissa Baker. The text designer was Eve Siegel. The cover designer was Mary McKeon. The index was prepared by Kathy Pitcoff. RR Donnelley was printer and binder. This book is printed on acid-free paper. ISBN-0-07-121766-5 (International Edition) Copyright © 2003. Exclusive rights by the McGraw-Hill Companies, Inc., for manufacture and export. This book cannot be re-exported from the country to which it is consigned by McGraw-Hill. The International Edition is not available in North America. fm01.qxd 3/16/04 11:10 AM Page vii Authors David A. Bender, PhD Peter A. Mayes, PhD, DSc Sub-Dean Royal Free and University College Medical Emeritus Professor of Veterinary Biochemistry, Royal School, Assistant Faculty Tutor and Tutor to Med- Veterinary College, University of London ical Students, Senior Lecturer in Biochemistry, De- partment of Biochemistry and Molecular Biology, University College London Robert K. Murray, MD, PhD Professor (Emeritus) of Biochemistry, University of Kathleen M. Botham, PhD, DSc Toronto Reader in Biochemistry, Royal Veterinary College, University of London Margaret L. Rand, PhD Scientist, Research Institute, Hospital for Sick Chil- Daryl K. Granner, MD dren, Toronto, and Associate Professor, Depart- ments of Laboratory Medicine and Pathobiology Joe C. Davis Professor of Biomedical Science, Director, and Department of Biochemistry, University of Vanderbilt Diabetes Center, Professor of Molecular Toronto Physiology and Biophysics and of Medicine, Vander- bilt University, Nashville, Tennessee Victor W. Rodwell, PhD Frederick W. Keeley, PhD Professor of Biochemistry, Purdue University, West Lafayette, Indiana Associate Director and Senior Scientist, Research Insti- tute, Hospital for Sick Children, Toronto, and Pro- fessor, Department of Biochemistry, University of P. Anthony Weil, PhD Toronto Professor of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nash- Peter J. Kennelly, PhD ville, Tennessee Professor of Biochemistry, Virginia Polytechnic Insti- tute and State University, Blacksburg, Virginia vii fm01.qxd 3/16/04 11:10 AM Page ix Preface The authors and publisher are pleased to present the twenty-sixth edition of Harper’s Illustrated Biochemistry. Review of Physiological Chemistry was first published in 1939 and revised in 1944, and it quickly gained a wide readership. In 1951, the third edition appeared with Harold A. Harper, University of California School of Medicine at San Fran- cisco, as author. Dr. Harper remained the sole author until the ninth edition and co-authored eight subsequent edi- tions. Peter Mayes and Victor Rodwell have been authors since the tenth edition, Daryl Granner since the twentieth edition, and Rob Murray since the twenty-first edition. Because of the increasing complexity of biochemical knowl- edge, they have added co-authors in recent editions. Fred Keeley and Margaret Rand have each co-authored one chapter with Rob Murray for this and previous edi- tions. Peter Kennelly joined as a co-author in the twenty-fifth edition, and in the present edition has co-authored with Victor Rodwell all of the chapters dealing with the structure and function of proteins and enzymes. The follow- ing additional co-authors are very warmly welcomed in this edition: Kathleen Botham has co-authored, with Peter Mayes, the chapters on bioenergetics, biologic oxidation, oxidative phosphorylation, and lipid metabolism. David Bender has co-authored, also with Peter Mayes, the chapters dealing with carbohydrate metabolism, nutrition, diges- tion, and vitamins and minerals. P. Anthony Weil has co-authored chapters dealing with various aspects of DNA, of RNA, and of gene expression with Daryl Granner. We are all very grateful to our co-authors for bringing their ex- pertise and fresh perspectives to the text. CHANGES IN THE TWENTY-SIXTH EDITION A major goal of the authors continues to be to provide both medical and other students of the health sciences with a book that both describes the basics of biochemistry and is user-friendly and interesting. A second major ongoing goal is to reflect the most significant advances in biochemistry that are important to medicine. However, a third major goal of this edition was to achieve a substantial reduction in size, as feedback indicated that many readers pre- fer shorter texts. To achieve this goal, all of the chapters were rigorously edited, involving their amalgamation, division, or dele- tion, and many were reduced to approximately one-half to two-thirds of their previous size. This has been effected without loss of crucial information but with gain in conciseness and clarity. Despite the reduction in size, there are many new features in the twenty-sixth edition. These include: A new chapter on amino acids and peptides, which emphasizes the manner in which the properties of biologic peptides derive from the individual amino acids of which they are comprised. A new chapter on the primary structure of proteins, which provides coverage of both classic and newly emerging “proteomic” and “genomic” methods for identifying proteins. A new section on the application of mass spectrometry to the analysis of protein structure has been added, including comments on the identification of covalent modifica- tions. The chapter on the mechanisms of action of enzymes has been revised to provide a comprehensive description of the various physical mechanisms by which enzymes carry out their catalytic functions. The chapters on integration of metabolism, nutrition, digestion and absorption, and vitamins and minerals have been completely re-written. Among important additions to the various chapters on metabolism are the following: update of the information on oxidative phosphorylation, including a description of the rotary ATP synthase; new insights into the role of GTP in gluconeogenesis; additional information on the regulation of acetyl-CoA carboxylase; new information on receptors involved in lipoprotein metabolism and reverse cholesterol transport; discussion of the role of leptin in fat storage; and new information on bile acid regulation, including the role of the farnesoid X receptor (FXR). The chapter on membrane biochemistry in the previous edition has been split into two, yielding two new chapters on the structure and function of membranes and intracellular traffic and sorting of proteins. Considerable new material has been added on RNA synthesis, protein synthesis, gene regulation, and various as- pects of molecular genetics. Much of the material on individual endocrine glands present in the twenty-fifth edition has been replaced with new chapters dealing with the diversity of the endocrine system, with molecular mechanisms of hormone action, and with signal transduction. ix fm01.qxd 3/16/04 11:10 AM Page x x / PREFACE The chapter on plasma proteins, immunoglobulins, and blood coagulation in the previous edition has been split into two new chapters on plasma proteins and immunoglobulins and on hemostasis and thrombosis. New information has been added in appropriate chapters on lipid rafts and caveolae, aquaporins, connexins, dis- orders due to mutations in genes encoding proteins involved in intracellular membrane transport, absorption of iron, and conformational diseases and pharmacogenomics. A new and final chapter on “The Human Genome Project” (HGP) has been added, which builds on the material covered in Chapters 35 through 40. Because of the impact of the results of the HGP on the future of biology and medicine, it appeared appropriate to conclude the text with a summary of its major findings and their implica- tions for future work. As initiated in the previous edition, references to useful Web sites have been included in a brief Appendix at the end of the text. ORGANIZATION OF THE BOOK The text is divided into two introductory chapters (“Biochemistry & Medicine” and “Water & pH”) followed by six main sections. Section I deals with the structures and functions of proteins and enzymes, the workhorses of the body. Because almost all of the reactions in cells are catalyzed by enzymes, it is vital to understand the properties of enzymes before considering other topics. Section II explains how various cellular reactions either utilize or release energy, and it traces the pathways by which carbohydrates and lipids are synthesized and degraded. It also describes the many functions of these two classes of molecules. Section III deals with the amino acids and their many fates and also describes certain key features of protein ca- tabolism. Section IV describes the structures and functions of the nucleotides and nucleic acids, and covers many major topics such as DNA replication and repair, RNA synthesis and modification, and protein synthesis. It also discusses new findings on how genes are regulated and presents the principles of recombinant DNA technology. Section V deals with aspects of extracellular and intracellular communication. Topics covered include membrane structure and function, the molecular bases of the actions of hormones, and the key field of signal transduction. Section VI consists of discussions of eleven special topics: nutrition, digestion, and absorption; vitamins and minerals; intracellular traffic and sorting of proteins; glycoproteins; the extracellular matrix; muscle and the cy- toskeleton; plasma proteins and immunoglobulins; hemostasis and thrombosis; red and white blood cells; the me- tabolism of xenobiotics; and the Human Genome Project. ACKNOWLEDGMENTS The authors thank Janet Foltin for her thoroughly professional approach. Her constant interest and input have had a significant impact on the final structure of this text. We are again immensely grateful to Jim Ransom for his excel- lent editorial work; it has been a pleasure to work with an individual who constantly offered wise and informed alter- natives to the sometimes primitive text transmitted by the authors. The superb editorial skills of Janene Matragrano Oransky and Harriet Lebowitz are warmly acknowledged, as is the excellent artwork of Charissa Baker and her col- leagues. The authors are very grateful to Kathy Pitcoff for her thoughtful and meticulous work in preparing the Index. Suggestions from students and colleagues around the world have been most helpful in the formulation of this edition. We look forward to receiving similar input in the future. Robert K. Murray, MD, PhD Daryl K. Granner, MD Peter A. Mayes, PhD, DSc Victor W. Rodwell, PhD Toronto, Ontario Nashville, Tennessee London West Lafayette, Indiana March 2003 fm01.qxd 3/16/04 11:10 AM Page iii Contents Authors............................................................................. vii Preface.............................................................................. ix 1. Biochemistry & Medicine Robert K. Murray, MD, PhD........................................................... 1 2. Water & pH Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD........................................... 5 SECTION I. STRUCTURES & FUNCTIONS OF PROTEINS & ENZYMES................... 14 3. Amino Acids & Peptides Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 14 4. Proteins: Determination of Primary Structure Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 21 5. Proteins: Higher Orders of Structure Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 30 6. Proteins: Myoglobin & Hemoglobin Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 40 7. Enzymes: Mechanism of Action Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 49 8. Enzymes: Kinetics Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 60 9. Enzymes: Regulation of Activities Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD.......................................... 72 SECTION II. BIOENERGETICS & THE METABOLISM OF CARBOHYDRATES & LIPIDS....................................................................... 80 10. Bioenergetics: The Role of ATP Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................. 80 11. Biologic Oxidation Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................. 86 12. The Respiratory Chain & Oxidative Phosphorylation Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................. 92 13. Carbohydrates of Physiologic Significance Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 102 iii fm01.qxd 3/16/04 11:10 AM Page iv iv / CONTENTS 14. Lipids of Physiologic Significance Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 111 15. Overview of Metabolism Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 122 16. The Citric Acid Cycle: The Catabolism of Acetyl-CoA Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 130 17. Glycolysis & the Oxidation of Pyruvate Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 136 18. Metabolism of Glycogen Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 145 19. Gluconeogenesis & Control of the Blood Glucose Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 153 20. The Pentose Phosphate Pathway & Other Pathways of Hexose Metabolism Peter A. Mayes, PhD, DSc, & David A. Bender, PhD....................................... 163 21. Biosynthesis of Fatty Acids Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 173 22. Oxidation of Fatty Acids: Ketogenesis Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 180 23. Metabolism of Unsaturated Fatty Acids & Eicosanoids Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 190 24. Metabolism of Acylglycerols & Sphingolipids Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 197 25. Lipid Transport & Storage Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 205 26. Cholesterol Synthesis, Transport, & Excretion Peter A. Mayes, PhD, DSc, & Kathleen M. Botham, PhD, DSc................................ 219 27. Integration of Metabolism—the Provision of Metabolic Fuels David A. Bender, PhD, & Peter A. Mayes, PhD, DSc....................................... 231 SECTION III. METABOLISM OF PROTEINS & AMINO ACIDS......................... 237 28. Biosynthesis of the Nutritionally Nonessential Amino Acids Victor W. Rodwell, PhD............................................................. 237 29. Catabolism of Proteins & of Amino Acid Nitrogen Victor W. Rodwell, PhD............................................................. 242 fm01.qxd 3/16/04 11:10 AM Page v CONTENTS / v 30. Catabolism of the Carbon Skeletons of Amino Acids Victor W. Rodwell, PhD............................................................. 249 31. Conversion of Amino Acids to Specialized Products Victor W. Rodwell, PhD............................................................. 264 32. Porphyrins & Bile Pigments Robert K. Murray, MD, PhD......................................................... 270 SECTION IV. STRUCTURE, FUNCTION, & REPLICATION OF INFORMATIONAL MACROMOLECULES........................................ 286 33. Nucleotides Victor W. Rodwell, PhD............................................................. 286 34. Metabolism of Purine & Pyrimidine Nucleotides Victor W. Rodwell, PhD............................................................. 293 35. Nucleic Acid Structure & Function Daryl K. Granner, MD............................................................. 303 36. DNA Organization, Replication, & Repair Daryl K. Granner, MD, & P. Anthony Weil, PhD......................................... 314 37. RNA Synthesis, Processing, & Modification Daryl K. Granner, MD, & P. Anthony Weil, PhD......................................... 341 38. Protein Synthesis & the Genetic Code Daryl K. Granner, MD............................................................. 358 39. Regulation of Gene Expression Daryl K. Granner, MD, & P. Anthony Weil, PhD......................................... 374 40. Molecular Genetics, Recombinant DNA, & Genomic Technology Daryl K. Granner, MD, & P. Anthony Weil, PhD......................................... 396 SECTION V. BIOCHEMISTRY OF EXTRACELLULAR & INTRACELLULAR COMMUNICATION........................................... 415 41. Membranes: Structure & Function Robert K. Murray, MD, PhD, & Daryl K. Granner, MD.................................... 415 42. The Diversity of the Endocrine System Daryl K. Granner, MD............................................................. 434 43. Hormone Action & Signal Transduction Daryl K. Granner, MD............................................................. 456 fm01.qxd 3/16/04 11:10 AM Page vi vi / CONTENTS SECTION VI. SPECIAL TOPICS.................................................... 474 44. Nutrition, Digestion, & Absorption David A. Bender, PhD, & Peter A. Mayes, PhD, DSc....................................... 474 45. Vitamins & Minerals David A. Bender, PhD, & Peter A. Mayes, PhD, DSc....................................... 481 46. Intracellular Traffic & Sorting of Proteins Robert K. Murray, MD, PhD......................................................... 498 47. Glycoproteins Robert K. Murray, MD, PhD......................................................... 514 48. The Extracellular Matrix Robert K. Murray, MD, PhD, & Frederick W. Keeley, PhD................................... 535 49. Muscle & the Cytoskeleton Robert K. Murray, MD, PhD......................................................... 556 50. Plasma Proteins & Immunoglobulins Robert K. Murray, MD, PhD......................................................... 580 51. Hemostasis & Thrombosis Margaret L. Rand, PhD, & Robert K. Murray, MD, PhD.................................... 598 52. Red & White Blood Cells Robert K. Murray, MD, PhD......................................................... 609 53. Metabolism of Xenobiotics Robert K. Murray, MD, PhD......................................................... 626 54. The Human Genome Project Robert K. Murray, MD, PhD......................................................... 633 Appendix.......................................................................... 639 Index.............................................................................. 643 ch01.qxd 2/13/2003 1:20 PM Page 1 Biochemistry & Medicine 1 Robert K. Murray, MD, PhD INTRODUCTION biochemistry is increasingly becoming their common language. Biochemistry can be defined as the science concerned with the chemical basis of life (Gk bios “life”). The cell is the structural unit of living systems. Thus, biochem- A Reciprocal Relationship Between istry can also be described as the science concerned with Biochemistry & Medicine Has Stimulated the chemical constituents of living cells and with the reac- Mutual Advances tions and processes they undergo. By this definition, bio- chemistry encompasses large areas of cell biology, of The two major concerns for workers in the health sci- molecular biology, and of molecular genetics. ences—and particularly physicians—are the understand- ing and maintenance of health and the understanding The Aim of Biochemistry Is to Describe & and effective treatment of diseases. Biochemistry im- pacts enormously on both of these fundamental con- Explain, in Molecular Terms, All Chemical cerns of medicine. In fact, the interrelationship of bio- Processes of Living Cells chemistry and medicine is a wide, two-way street. The major objective of biochemistry is the complete Biochemical studies have illuminated many aspects of understanding, at the molecular level, of all of the health and disease, and conversely, the study of various chemical processes associated with living cells. To aspects of health and disease has opened up new areas achieve this objective, biochemists have sought to iso- of biochemistry. Some examples of this two-way street late the numerous molecules found in cells, determine are shown in Figure 1–1. For instance, a knowledge of their structures, and analyze how they function. Many protein structure and function was necessary to eluci- techniques have been used for these purposes; some of date the single biochemical difference between normal them are summarized in Table 1–1. hemoglobin and sickle cell hemoglobin. On the other hand, analysis of sickle cell hemoglobin has contributed A Knowledge of Biochemistry Is Essential significantly to our understanding of the structure and to All Life Sciences function of both normal hemoglobin and other pro- teins. Analogous examples of reciprocal benefit between The biochemistry of the nucleic acids lies at the heart of biochemistry and medicine could be cited for the other genetics; in turn, the use of genetic approaches has been paired items shown in Figure 1–1. Another example is critical for elucidating many areas of biochemistry. the pioneering work of Archibald Garrod, a physician Physiology, the study of body function, overlaps with in England during the early 1900s. He studied patients biochemistry almost completely. Immunology employs with a number of relatively rare disorders (alkap- numerous biochemical techniques, and many immuno- tonuria, albinism, cystinuria, and pentosuria; these are logic approaches have found wide use by biochemists. described in later chapters) and established that these Pharmacology and pharmacy rest on a sound knowl- conditions were genetically determined. Garrod desig- edge of biochemistry and physiology; in particular, nated these conditions as inborn errors of metabo- most drugs are metabolized by enzyme-catalyzed reac- lism. His insights provided a major foundation for the tions. Poisons act on biochemical reactions or processes; development of the field of human biochemical genet- this is the subject matter of toxicology. Biochemical ap- ics. More recent efforts to understand the basis of the proaches are being used increasingly to study basic as- genetic disease known as familial hypercholesterol- pects of pathology (the study of disease), such as in- emia, which results in severe atherosclerosis at an early flammation, cell injury, and cancer. Many workers in age, have led to dramatic progress in understanding of microbiology, zoology, and botany employ biochemical cell receptors and of mechanisms of uptake of choles- approaches almost exclusively. These relationships are terol into cells. Studies of oncogenes in cancer cells not surprising, because life as we know it depends on have directed attention to the molecular mechanisms biochemical reactions and processes. In fact, the old involved in the control of normal cell growth. These barriers among the life sciences are breaking down, and and many other examples emphasize how the study of 1 ch01.qxd 2/13/2003 1:20 PM Page 2 2 / CHAPTER 1 Table 1–1. The principal methods and NORMAL BIOCHEMICAL PROCESSES ARE preparations used in biochemical laboratories. THE BASIS OF HEALTH The World Health Organization (WHO) defines Methods for Separating and Purifying Biomolecules1 health as a state of “complete physical, mental and so- Salt fractionation (eg, precipitation of proteins with ammo- cial well-being and not merely the absence of disease nium sulfate) Chromatography: Paper; ion exchange; affinity; thin-layer; and infirmity.” From a strictly biochemical viewpoint, gas-liquid; high-pressure liquid; gel filtration health may be considered that situation in which all of Electrophoresis: Paper; high-voltage; agarose; cellulose the many thousands of intra- and extracellular reactions acetate; starch gel; polyacrylamide gel; SDS-polyacryl- that occur in the body are proceeding at rates commen- amide gel surate with the organism’s maximal survival in the Ultracentrifugation physiologic state. However, this is an extremely reduc- Methods for Determining Biomolecular Structures tionist view, and it should be apparent that caring for Elemental analysis the health of patients requires not only a wide knowl- UV, visible, infrared, and NMR spectroscopy edge of biologic principles but also of psychologic and Use of acid or alkaline hydrolysis to degrade the biomole- social principles. cule under study into its basic constituents Use of a battery of enzymes of known specificity to de- grade the biomolecule under study (eg, proteases, nucle- Biochemical Research Has Impact on ases, glycosidases) Nutrition & Preventive Medicine Mass spectrometry One major prerequisite for the maintenance of health is Specific sequencing methods (eg, for proteins and nucleic that there be optimal dietary intake of a number of acids) chemicals; the chief of these are vitamins, certain X-ray crystallography amino acids, certain fatty acids, various minerals, and Preparations for Studying Biochemical Processes Whole animal (includes transgenic animals and animals water. Because much of the subject matter of both bio- with gene knockouts) chemistry and nutrition is concerned with the study of Isolated perfused organ various aspects of these chemicals, there is a close rela- Tissue slice tionship between these two sciences. Moreover, more Whole cells emphasis is being placed on systematic attempts to Homogenate maintain health and forestall disease, ie, on preventive Isolated cell organelles medicine. Thus, nutritional approaches to—for exam- Subfractionation of organelles ple—the prevention of atherosclerosis and cancer are Purified metabolites and enzymes receiving increased emphasis. Understanding nutrition Isolated genes (including polymerase chain reaction and depends to a great extent on a knowledge of biochem- site-directed mutagenesis) istry. 1 Most of these methods are suitable for analyzing the compo- nents present in cell homogenates and other biochemical prepa- Most & Perhaps All Disease Has rations. The sequential use of several techniques will generally permit purification of most biomolecules. The reader is referred a Biochemical Basis to texts on methods of biochemical research for details. We believe that most if not all diseases are manifesta- tions of abnormalities of molecules, chemical reactions, or biochemical processes. The major factors responsible disease can open up areas of cell function for basic bio- for causing diseases in animals and humans are listed in chemical research. Table 1–2. All of them affect one or more critical The relationship between medicine and biochem- chemical reactions or molecules in the body. Numerous istry has important implications for the former. As long examples of the biochemical bases of diseases will be en- as medical treatment is firmly grounded in a knowledge countered in this text; the majority of them are due to of biochemistry and other basic sciences, the practice of causes 5, 7, and 8. In most of these conditions, bio- medicine will have a rational basis that can be adapted chemical studies contribute to both the diagnosis and to accommodate new knowledge. This contrasts with treatment. Some major uses of biochemical investiga- unorthodox health cults and at least some “alternative tions and of laboratory tests in relation to diseases are medicine” practices, which are often founded on little summarized in Table 1–3. more than myth and wishful thinking and generally Additional examples of many of these uses are pre- lack any intellectual basis. sented in various sections of this text. ch01.qxd 2/13/2003 1:20 PM Page 3 BIOCHEMISTRY & MEDICINE / 3 BIOCHEMISTRY Nucleic acids Proteins Lipids Carbohydrates Genetic Sickle cell Athero- Diabetes diseases anemia sclerosis mellitus MEDICINE Figure 1–1. Examples of the two-way street connecting biochemistry and medicine. Knowledge of the biochemical molecules shown in the top part of the diagram has clarified our understanding of the diseases shown in the bottom half—and conversely, analyses of the diseases shown below have cast light on many areas of biochemistry. Note that sickle cell anemia is a genetic disease and that both atherosclerosis and diabetes mellitus have genetic components. Impact of the Human Genome Project (HGP) on Biochemistry & Medicine Table 1–3. Some uses of biochemical Remarkable progress was made in the late 1990s in se- quencing the human genome. This culminated in July investigations and laboratory tests in 2000, when leaders of the two groups involved in this relation to diseases. effort (the International Human Genome Sequencing Consortium and Celera Genomics, a private company) Use Example announced that over 90% of the genome had been se- 1. To reveal the funda- Demonstration of the na- quenced. Draft versions of the sequence were published mental causes and ture of the genetic de- mechanisms of diseases fects in cystic fibrosis. 2. To suggest rational treat- A diet low in phenylalanine Table 1–2. The major causes of diseases. All of ments of diseases based for treatment of phenyl- the causes listed act by influencing the various on (1) above ketonuria. biochemical mechanisms in the cell or in the 3. To assist in the diagnosis Use of the plasma enzyme of specific diseases creatine kinase MB body.1 (CK-MB) in the diagnosis of myocardial infarction. 1. Physical agents: Mechanical trauma, extremes of temper- 4. To act as screening tests Use of measurement of ature, sudden changes in atmospheric pressure, radia- for the early diagnosis blood thyroxine or tion, electric shock. of certain diseases thyroid-stimulating hor- 2. Chemical agents, including drugs: Certain toxic com- mone (TSH) in the neo- pounds, therapeutic drugs, etc. natal diagnosis of con- 3. Biologic agents: Viruses, bacteria, fungi, higher forms of genital hypothyroidism. parasites. 5. To assist in monitoring Use of the plasma enzyme 4. Oxygen lack: Loss of blood supply, depletion of the the progress (eg, re- alanine aminotransferase oxygen-carrying capacity of the blood, poisoning of covery, worsening, re- (ALT) in monitoring the the oxidative enzymes. mission, or relapse) of progress of infectious 5. Genetic disorders: Congenital, molecular. certain diseases hepatitis. 6. Immunologic reactions: Anaphylaxis, autoimmune 6. To assist in assessing Use of measurement of disease. the response of dis- blood carcinoembryonic 7. Nutritional imbalances: Deficiencies, excesses. eases to therapy antigen (CEA) in certain 8. Endocrine imbalances: Hormonal deficiencies, excesses. patients who have been 1 treated for cancer of the Adapted, with permission, from Robbins SL, Cotram RS, Kumar V: The Pathologic Basis of Disease, 3rd ed. Saunders, 1984. colon. ch01.qxd 2/13/2003 1:20 PM Page 4 4 / CHAPTER 1 in early 2001. It is anticipated that the entire sequence The judicious use of various biochemical laboratory will be completed by 2003. The implications of this tests is an integral component of diagnosis and moni- work for biochemistry, all of biology, and for medicine toring of treatment. are tremendous, and only a few points are mentioned A sound knowledge of biochemistry and of other re- here. Many previously unknown genes have been re- lated basic disciplines is essential for the rational vealed; their protein products await characterization. practice of medical and related health sciences. New light has been thrown on human evolution, and procedures for tracking disease genes have been greatly refined. The results are having major effects on areas REFERENCES such as proteomics, bioinformatics, biotechnology, and Fruton JS: Proteins, Enzymes, Genes: The Interplay of Chemistry and pharmacogenomics. Reference to the human genome Biology. Yale Univ Press, 1999. (Provides the historical back- will be made in various sections of this text. The ground for much of today’s biochemical research.) Human Genome Project is discussed in more detail in Garrod AE: Inborn errors of metabolism. (Croonian Lectures.) Chapter 54. Lancet 1908;2:1, 73, 142, 214. International Human Genome Sequencing Consortium. Initial se- SUMMARY quencing and analysis of the human genome. Nature 2001:409;860. (The issue [15 February] consists of articles Biochemistry is the science concerned with studying dedicated to analyses of the human genome.) the various molecules that occur in living cells and Kornberg A: Basic research: The lifeline of medicine. FASEB J organisms and with their chemical reactions. Because 1992;6:3143. life depends on biochemical reactions, biochemistry Kornberg A: Centenary of the birth of modern biochemistry. has become the basic language of all biologic sci- FASEB J 1997;11:1209. ences. McKusick VA: Mendelian Inheritance in Man. Catalogs of Human Genes and Genetic Disorders, 12th ed. Johns Hopkins Univ Biochemistry is concerned with the entire spectrum Press, 1998. [Abbreviated MIM] of life forms, from relatively simple viruses and bacte- Online Mendelian Inheritance in Man (OMIM): Center for Med- ria to complex human beings. ical Genetics, Johns Hopkins University and National Center Biochemistry and medicine are intimately related. for Biotechnology Information, National Library of Medi- Health depends on a harmonious balance of bio- cine, 1997. http://www.ncbi.nlm.nih.gov/omim/ chemical reactions occurring in the body, and disease (The numbers assigned to the entries in MIM and OMIM will be reflects abnormalities in biomolecules, biochemical cited in selected chapters of this work. Consulting this exten- sive collection of diseases and other relevant entries—specific reactions, or biochemical processes. proteins, enzymes, etc—will greatly expand the reader’s Advances in biochemical knowledge have illumi- knowledge and understanding of various topics referred to nated many areas of medicine. Conversely, the study and discussed in this text. The online version is updated al- of diseases has often revealed previously unsuspected most daily.) aspects of biochemistry. The determination of the se- Scriver CR et al (editors): The Metabolic and Molecular Bases of In- quence of the human genome, nearly complete, will herited Disease, 8th ed. McGraw-Hill, 2001. have a great impact on all areas of biology, including Venter JC et al: The Sequence of the Human Genome. Science 2001;291:1304. (The issue [16 February] contains the Celera biochemistry, bioinformatics, and biotechnology. draft version and other articles dedicated to analyses of the Biochemical approaches are often fundamental in il- human genome.) luminating the causes of diseases and in designing Williams DL, Marks V: Scientific Foundations of Biochemistry in appropriate therapies. Clinical Practice, 2nd ed. Butterworth-Heinemann, 1994. ch02.qxd 2/13/2003 1:41 PM Page 5 Water & pH 2 Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD BIOMEDICAL IMPORTANCE oxygen atom pulls electrons away from the hydrogen nuclei, leaving them with a partial positive charge, Water is the predominant chemical component of liv- while its two unshared electron pairs constitute a region ing organisms. Its unique physical properties, which in- of local negative charge. clude the ability to solvate a wide range of organic and Water, a strong dipole, has a high dielectric con- inorganic molecules, derive from water’s dipolar struc- stant. As described quantitatively by Coulomb’s law, ture and exceptional capacity for forming hydrogen the strength of interaction F between oppositely bonds. The manner in which water interacts with a sol- charged particles is inversely proportionate to the di- vated biomolecule influences the structure of each. An electric constant ε of the surrounding medium. The di- excellent nucleophile, water is a reactant or product in electric constant for a vacuum is unity; for hexane it is many metabolic reactions. Water has a slight propensity 1.9; for ethanol it is 24.3; and for water it is 78.5. to dissociate into hydroxide ions and protons. The Water therefore greatly decreases the force of attraction acidity of aqueous solutions is generally reported using between charged and polar species relative to water-free the logarithmic pH scale. Bicarbonate and other buffers environments with lower dielectric constants. Its strong normally maintain the pH of extracellular fluid be- dipole and high dielectric constant enable water to dis- tween 7.35 and 7.45. Suspected disturbances of acid- solve large quantities of charged compounds such as base balance are verified by measuring the pH of arter- salts. ial blood and the CO2 content of venous blood. Causes of acidosis (blood pH < 7.35) include diabetic ketosis and lactic acidosis. Alkalosis (pH > 7.45) may, for ex- Water Molecules Form Hydrogen Bonds ample, follow vomiting of acidic gastric contents. Regu- lation of water balance depends upon hypothalamic An unshielded hydrogen nucleus covalently bound to mechanisms that control thirst, on antidiuretic hor- an electron-withdrawing oxygen or nitrogen atom can mone (ADH), on retention or excretion of water by the interact with an unshared electron pair on another oxy- kidneys, and on evaporative loss. Nephrogenic diabetes gen or nitrogen atom to form a hydrogen bond. Since insipidus, which involves the inability to concentrate water molecules contain both of these features, hydro- urine or adjust to subtle changes in extracellular fluid gen bonding favors the self-association of water mole- osmolarity, results from the unresponsiveness of renal cules into ordered arrays (Figure 2–2). Hydrogen bond- tubular osmoreceptors to ADH. ing profoundly influences the physical properties of water and accounts for its exceptionally high viscosity, surface tension, and boiling point. On average, each molecule in liquid water associates through hydrogen WATER IS AN IDEAL BIOLOGIC SOLVENT bonds with 3.5 others. These bonds are both relatively Water Molecules Form Dipoles weak and transient, with a half-life of about one mi- crosecond. Rupture of a hydrogen bond in liquid water A water molecule is an irregular, slightly skewed tetra- requires only about 4.5 kcal/mol, less than 5% of the hedron with oxygen at its center (Figure 2–1). The two energy required to rupture a covalent O H bond. hydrogens and the unshared electrons of the remaining Hydrogen bonding enables water to dissolve many two sp3-hybridized orbitals occupy the corners of the organic biomolecules that contain functional groups tetrahedron. The 105-degree angle between the hydro- which can participate in hydrogen bonding. The oxy- gens differs slightly from the ideal tetrahedral angle, gen atoms of aldehydes, ketones, and amides provide 109.5 degrees. Ammonia is also tetrahedral, with a 107- pairs of electrons that can serve as hydrogen acceptors. degree angle between its hydrogens. Water is a dipole, Alcohols and amines can serve both as hydrogen accep- a molecule with electrical charge distributed asymmetri- tors and as donors of unshielded hydrogen atoms for cally about its structure. The strongly electronegative formation of hydrogen bonds (Figure 2–3). 5 ch02.qxd 2/13/2003 1:41 PM Page 6 6 / CHAPTER 2 H CH3 CH2 O H O 2e H 2e H H 105° CH3 CH2 O H O H CH2 CH3 Figure 2–1. The water molecule has tetrahedral R R II geometry. C O H N RI R III INTERACTION WITH WATER INFLUENCES THE STRUCTURE OF BIOMOLECULES Figure 2–3. Additional polar groups participate in hydrogen bonding. Shown are hydrogen bonds formed Covalent & Noncovalent Bonds Stabilize between an alcohol and water, between two molecules Biologic Molecules of ethanol, and between the peptide carbonyl oxygen The covalent bond is the strongest force that holds and the peptide nitrogen hydrogen of an adjacent molecules together (Table 2–1). Noncovalent forces, amino acid. while of lesser magnitude, make significant contribu- tions to the structure, stability, and functional compe- phosphatidyl serine or phosphatidyl ethanolamine con- tence of macromolecules in living cells. These forces, tact water while their hydrophobic fatty acyl side chains which can be either attractive or repulsive, involve in- cluster together, excluding water. This pattern maxi- teractions both within the biomolecule and between it mizes the opportunities for the formation of energeti- and the water that forms the principal component of cally favorable charge-dipole, dipole-dipole, and hydro- the surrounding environment. gen bonding interactions between polar groups on the biomolecule and water. It also minimizes energetically Biomolecules Fold to Position Polar & unfavorable contact between water and hydrophobic Charged Groups on Their Surfaces groups. Most biomolecules are amphipathic; that is, they pos- sess regions rich in charged or polar functional groups Hydrophobic Interactions as well as regions with hydrophobic character. Proteins Hydrophobic interaction refers to the tendency of non- tend to fold with the R-groups of amino acids with hy- polar compounds to self-associate in an aqueous envi- drophobic side chains in the interior. Amino acids with ronment. This self-association is driven neither by mu- charged or polar amino acid side chains (eg, arginine, tual attraction nor by what are sometimes incorrectly glutamate, serine) generally are present on the surface referred to as “hydrophobic bonds.” Self-association in contact with water. A similar pattern prevails in a arises from the need to minimize energetically unfavor- phospholipid bilayer, where the charged head groups of able interactions between nonpolar groups and water. H H H H Table 2–1. Bond energies for atoms of biologic O O significance. H H H H O O H O Bond Energy Bond Energy H O H H Type (kcal/mol) Type (kcal/mol) H O H O—O 34 O==O 96 S—S 51 C—H 99 Figure 2–2. Left: Association of two dipolar water C—N 70 C==S 108 molecules by a hydrogen bond (dotted line). Right: S—H 81 O—H 110 Hydrogen-bonded cluster of four water molecules. C—C 82 C==C 147 C—O 84 C==N 147 Note that water can serve simultaneously both as a hy- N—H 94 C==O 164 drogen donor and as a hydrogen acceptor. ch02.qxd 2/13/2003 1:41 PM Page 7 WATER & pH / 7 While the hydrogens of nonpolar groups such as the the backbone to water while burying the relatively hy- methylene groups of hydrocarbons do not form hydro- drophobic nucleotide bases inside. The extended back- gen bonds, they do affect the structure of the water that bone maximizes the distance between negatively surrounds them. Water molecules adjacent to a hy- charged backbone phosphates, minimizing unfavorable drophobic group are restricted in the number of orien- electrostatic interactions. tations (degrees of freedom) that permit them to par- ticipate in the maximum number of energetically WATER IS AN EXCELLENT NUCLEOPHILE favorable hydrogen bonds. Maximal formation of mul- tiple hydrogen bonds can be maintained only by in- Metabolic reactions often involve the attack by lone creasing the order of the adjacent water molecules, with pairs of electrons on electron-rich molecules termed a corresponding decrease in entropy. nucleophiles on electron-poor atoms called elec- It follows from the second law of thermodynamics trophiles. Nucleophiles and electrophiles do not neces- that the optimal free energy of a hydrocarbon-water sarily possess a formal negative or positive charge. mixture is a function of both maximal enthalpy (from Water, whose two lone pairs of sp3 electrons bear a par- hydrogen bonding) and minimum entropy (maximum tial negative charge, is an excellent nucleophile. Other degrees of freedom). Thus, nonpolar molecules tend to nucleophiles of biologic importance include the oxygen form droplets with minimal exposed surface area, re- atoms of phosphates, alcohols, and carboxylic acids; the ducing the number of water molecules affected. For the sulfur of thiols; the nitrogen of amines; and the imid- same reason, in the aqueous environment of the living azole ring of histidine. Common electrophiles include cell the hydrophobic portions of biopolymers tend to the carbonyl carbons in amides, esters, aldehydes, and be buried inside the structure of the molecule, or within ketones and the phosphorus atoms of phosphoesters. a lipid bilayer, minimizing contact with water. Nucleophilic attack by water generally results in the cleavage of the amide, glycoside, or ester bonds that hold biopolymers together. This process is termed hy- Electrostatic Interactions drolysis. Conversely, when monomer units are joined Interactions between charged groups shape biomolecu- together to form biopolymers such as proteins or glyco- lar structure. Electrostatic interactions between oppo- gen, water is a product, as shown below for the forma- sitely charged groups within or between biomolecules tion of a peptide bond between two amino acids. are termed salt bridges. Salt bridges are comparable in strength to hydrogen bonds but act over larger dis- O + tances. They thus often facilitate the binding of charged H3N OH + H NH molecules and ions to proteins and nucleic acids. O– Alanine Van der Waals Forces O Van der Waals forces arise from attractions between Valine transient dipoles generated by the rapid movement of electrons on all neutral atoms. Significantly weaker than hydrogen bonds but potentially extremely numer- H2O ous, van der Waals forces decrease as the sixth power of the distance separating atoms. Thus, they act over very O + short distances, typically 2–4 Å. H3 N NH Multiple Forces Stabilize Biomolecules O– The DNA double helix illustrates the contribution of O multiple forces to the structure of biomolecules. While each individual DNA strand is held together by cova- While hydrolysis is a thermodynamically favored re- lent bonds, the two strands of the helix are held to- action, the amide and phosphoester bonds of polypep- gether exclusively by noncovalent interactions. These tides and oligonucleotides are stable in the aqueous en- noncovalent interactions include hydrogen bonds be- vironment of the cell. This seemingly paradoxic tween nucleotide bases (Watson-Crick base pairing) behavior reflects the fact that the thermodynamics gov- and van der Waals interactions between the stacked erning the equilibrium of a reaction do not determine purine and pyrimidine bases. The helix presents the the rate at which it will take place. In the cell, protein charged phosphate groups and polar ribose sugars of catalysts called enzymes are used to accelerate the rate ch02.qxd 2/13/2003 1:41 PM Page 8 8 / CHAPTER 2 of hydrolytic reactions when needed. Proteases catalyze H7O3+. The proton is nevertheless routinely repre- the hydrolysis of proteins into their component amino sented as H+, even though it is in fact highly hydrated. acids, while nucleases catalyze the hydrolysis of the Since hydronium and hydroxide ions continuously phosphoester bonds in DNA and RNA. Careful control recombine to form water molecules, an individual hy- of the activities of these enzymes is required to ensure drogen or oxygen cannot be stated to be present as an that they act only on appropriate target molecules. ion or as part of a water molecule. At one instant it is an ion. An instant later it is part of a molecule. Individ- Many Metabolic Reactions Involve ual ions or molecules are therefore not considered. We Group Transfer refer instead to the probability that at any instant in time a hydrogen will be present as an ion or as part of a In group transfer reactions, a group G is transferred water molecule. Since 1 g of water contains 3.46 × 1022 from a donor D to an acceptor A, forming an acceptor molecules, the ionization of water can be described sta- group complex A–G: tistically. To state that the probability that a hydrogen exists as an ion is 0.01 means that a hydrogen atom has D−G + A = A−G + D one chance in 100 of being an ion and 99 chances out The hydrolysis and phosphorolysis of glycogen repre- of 100 of being part of a water molecule. The actual sent group transfer reactions in which glucosyl groups probability of a hydrogen atom in pure water existing as are transferred to water or to orthophosphate. The a hydrogen ion is approximately 1.8 × 10−9. The proba- equilibrium constant for the hydrolysis of covalent bility of its being part of a molecule thus is almost bonds strongly favors the formation of split products. unity. Stated another way, for every hydrogen ion and The biosynthesis of macromolecules also involves group hydroxyl ion in pure water there are 1.8 billion or 1.8 × transfer reactions in which the thermodynamically un- 109 water molecules. Hydrogen ions and hydroxyl ions favored synthesis of covalent bonds is coupled to fa- nevertheless contribute significantly to the properties of vored reactions so that the overall change in free energy water. favors biopolymer synthesis. Given the nucleophilic For dissociation of water, character of water and its high concentration in cells, why are biopolymers such as proteins and DNA rela- [H+ ][OH− ] K= tively stable? And how can synthesis of biopolymers [H2O] occur in an apparently aqueous environment? Central to both questions are the properties of enzymes. In the where brackets represent molar concentrations (strictly absence of enzymic catalysis, even thermodynamically speaking, molar activities) and K is the dissociation highly favored reactions do not necessarily take place constant. Since one mole (mol) of water weighs 18 g, rapidly. Precise and differential control of enzyme ac- one liter (L) (1000 g) of water contains 1000 × 18 = tivity and the sequestration of enzymes in specific or- 55.56 mol. Pure water thus is 55.56 molar. Since the ganelles determine under what physiologic conditions a probability that a hydrogen in pure water will exist as a given biopolymer will be synthesized or degraded. hydrogen ion is 1.8 × 10−9, the molar concentration of Newly synthesized polymers are not immediately hy- H+ ions (or of OH− ions) in pure water is the product drolyzed, in part because the active sites of biosynthetic of the probability, 1.8 × 10−9, times the molar concen- enzymes sequester substrates in an environment from tration of water, 55.56 mol/L. The result is 1.0 × 10−7 which water can be excluded. mol/L. We can now calculate K for water: Water Molecules Exhibit a Slight but Important Tendency to Dissociate [H + ][ OH − ] [10 −7 ][10 −7 ] K= = The ability of water to ionize, while slight, is of central [H2 O ] [ 55.56 ] importance for life. Since water can act both as an acid and as a base, its ionization may be represented as an = 0.018 × 10 −14 = 1.8 × 10 −16 mol / L intermolecular proton transfer that forms a hydronium ion (H3O+) and a hydroxide ion (OH−): The molar concentration of water, 55.56 mol/L, is too great to be significantly affected by dissociation. It H2O + H2O =H3O+ + OH− therefore is considered to be essentially constant. This constant may then be incorporated into the dissociation The transferred proton is actually associated with a constant K to provide a useful new constant Kw termed cluster of water molecules. Protons exist in solution not the ion product for water. The relationship between only as H3O+, but also as multimers such as H5O2+ and Kw and K is shown below: ch02.qxd 2/13/2003 1:41 PM Page 9 WATER & pH / 9 [H + ][ OH − ] termediates, whose phosphoryl group contains two dis- K= = 1.8 × 10 −16 mol / L sociable protons, the first of which is strongly acidic. [H2 O ] The following examples illustrate how to calculate K w = (K )[H2 O ] = [H + ][ OH − ] the pH of acidic and basic solutions. Example 1: What is the pH of a solution whose hy- = (1.8 × 10 −16 mol / L ) ( 55.56 mol / L ) drogen ion concentration is 3.2 × 10− 4 mol/L? = 1.00 × 10 −14 (mol / L )2 pH = − log [H+ ] Note that the dimensions of K are moles per liter and = − log (3.2 × 10 − 4 ) those of Kw are moles2 per liter2. As its name suggests, = − log (3.2) − log (10 − 4 ) the ion product Kw is numerically equal to the product of the molar concentrations of H+ and OH−: = −0.5 + 4.0 = 3.5 K w = [H + ][ OH − ] Example 2: What is the pH of a solution whose hy- At 25 °C, Kw = (10−7)2, or 10−14 (mol/L)2. At tempera- droxide ion concentration is 4.0 × 10− 4 mol/L? We first tures below 25 °C, Kw is somewhat less than 10−14; and define a quantity pOH that is equal to −log [OH−] and at temperatures above 25 °C it is somewhat greater than that may be derived from the definition of Kw: 10−14. Within the stated limitations of the effect of tem- perature, Kw equals 10-14 (mol/L)2 for all aqueous so- K w = [H + ][ OH − ] = 10 −14 lutions, even solutions of acids or bases. We shall use Kw to calculate the pH of acidic and basic solutions. Therefore: pH IS THE NEGATIVE LOG OF THE log [H + ] + log [ OH − ] = log 10 −14 HYDROGEN ION CONCENTRATION or The term pH was introduced in 1909 by Sörensen, who defined pH as the negative log of the hydrogen ion pH + pOH = 14 concentration: To solve the problem by this approach: pH = −log [H + ] [OH− ] = 4.0 × 10 − 4 This definition, while not rigorous, suffices for many biochemical purposes. To calculate the pH of a solution: pOH = − log [OH− ] 1. Calculate hydrogen ion concentration [H+]. = − log (4.0 × 10 − 4 ) 2. Calculate the base 10 logarithm of [H+]. = − log (4.0) − log (10 − 4 ) 3. pH is the negative of the value found in step 2. = −0.60 + 4.0 For example, for pure water at 25°C, = 3.4 pH = − log [H + ] = −log 10 −7 = −( −7) = 7.0 Now: Low pH values correspond to high concentrations of pH = 14 − pOH = 14 − 3.4 H+ and high pH values correspond to low concentra- = 10.6 tions of H+. Acids are proton donors and bases are proton ac- Example 3: What are the pH values of (a) 2.0 × 10−2 ceptors. Strong acids (eg, HCl or H2SO4) completely mol/L KOH and of (b) 2.0 × 10−6 mol/L KOH? The dissociate into anions and cations even in strongly acidic OH− arises from two sources, KOH and water. Since solutions (low pH). Weak acids dissociate only partially pH is determined by the total [H+] (and pOH by the in acidic solutions. Similarly, strong bases (eg, KOH or total [OH−]), both sources must be considered. In the NaOH)—but not weak bases (eg, Ca[OH]2)—are first case (a), the contribution of water to the total completely dissociated at high pH. Many biochemicals [OH−] is negligible. The same cannot be said for the are weak acids. Exceptions include phosphorylated in- second case (b): ch02.qxd 2/13/2003 1:41 PM Page 10 10 / CHAPTER 2 Concentration (mol/L) below are the expressions for the dissociation constant (Ka ) for two representative weak acids, RCOOH and (a) (b) RNH3+. −2 Molarity of KOH 2.0 × 10 2.0 × 10−6 [OH−] from KOH 2.0 × 10−2 2.0 × 10−6 R — COOH =R — COO− + H+ [OH−] from water 1.0 × 10−7 1.0 × 10−7 [R — COO− ][H+ ] Total [OH−] 2.00001 × 10−2 2.1 × 10−6 Ka = [R — COOH] R — NH3+ =R — NH2 + H+ Once a decision has been reached about the significance of the contribution by water, pH may be calculated as [R — NH2 ][H+ ] Ka = above. [R — NH3+ ] The above examples assume that the strong base KOH is completely dissociated in solution and that the Since the numeric values of Ka for weak acids are nega- concentration of OH− ions was thus equal to that of the tive exponential numbers, we express Ka as pKa, where KOH. This assumption is valid for dilute solutions of strong bases or acids but not for weak bases or acids. pK a = − log K Since weak electrolytes dissociate only slightly in solu- tion, we must use the dissociation constant to calcu- Note that pKa is related to Ka as pH is to [H+]. The late the concentration of [H+] (or [OH−]) produced by stronger the acid, the lower its pKa value. a given molarity of a weak acid (or base) before calcu- pKa is used to express the relative strengths of both lating total [H+] (or total [OH−]) and subsequently pH. acids and bases. For any weak acid, its conjugate is a strong base. Similarly, the conjugate of a strong base is a weak acid. The relative strengths of bases are ex- Functional Groups That Are Weak Acids pressed in terms of the pKa of their conjugate acids. For Have Great Physiologic Significance polyproteic compounds containing more than one dis- Many biochemicals possess functional groups that are sociable proton, a numerical subscript is assigned to weak acids or bases. Carboxyl groups, amino groups, each in order of relative acidity. For a dissociation of and the second phosphate dissociation of phosphate es- the type ters are present in proteins and nucleic acids, most + coenzymes, and most intermediary metabolites. Knowl- R — NH3 → R — NH2 edge of the dissociation of weak acids and bases thus is basic to understanding the influence of intracellular pH the pKa is the pH at which the concentration of the on structure and biologic activity. Charge-based separa- acid RNH3+ equals that of the base RNH2. tions such as electrophoresis and ion exchange chro- From the above equations that relate Ka to [H+] and matography also are best understood in terms of the to the concentrations of undissociated acid and its con- dissociation behavior of functional groups. jugate base, when We term the protonated species (eg, HA or RNH3+) the acid and the unprotonated species (eg, [R — COO− ] = [R — COOH] A− or RNH2) its conjugate base. Similarly, we may refer to a base (eg, A− or RNH2) and its conjugate acid (eg, HA or RNH3+). Representative weak acids or when (left), their conjugate bases (center), and the pKa values (right) include the following: [R — NH2 ] = [R — NH3 + ] R — CH2 — COOH R — CH2 — COO− pK a = 4 − 5 then + R — CH2 — NH3 R — CH2 — NH2 pK a = 9 − 10 K a = [H+ ] − H2CO3 HCO3 pK a = 6.4 − −2 Thus, when the associated (protonated) and dissociated H2PO4 HPO4 pK a = 7.2 (conjugate base) species are present at equal concentra- tions, the prevailing hydrogen ion concentration [H+] We express the relative strengths of weak acids and is numerically equal to the dissociation constant, Ka. If bases in terms of their dissociation constants. Shown the logarithms of both sides of the above equation are ch02.qxd 2/13/2003 1:41 PM Page 11 WATER & pH / 11 taken and both sides are multiplied by −1, the expres- Substitute pH and pKa for −log [H+] and −log Ka, re- sions would be as follows: spectively; then: K a = [H+ ] [HA ] pH = pK a − log − log K a = −log [H+ ] [A − ] Since −log Ka is defined as pKa, and −log [H+] de- Inversion of the last term removes the minus sign fines pH, the equation may be rewritten as and gives the Henderson-Hasselbalch equation: pK a = pH [A − ] pH = pK a + log [HA ] ie, the pKa of an acid group is the pH at which the pro- tonated and unprotonated species are present at equal concentrations. The pKa for an acid may be determined The Henderson-Hasselbalch equation has great pre- by adding 0.5 equivalent of alkali per equivalent of dictive value in protonic equilibria. For example, acid. The resulting pH will be the pKa of the acid. (1) When an acid is exactly half-neutralized, [A−] = [HA]. Under these conditions, The Henderson-Hasselbalch Equation [A − ] 1 Describes the Behavior pH = pK a + log = pK a + log = pK a + 0 [HA ] 1 of Weak Acids & Buffers The Henderson-Hasselbalch equation is derived below. Therefore, at half-neutralization, pH = pKa. A weak acid, HA, ionizes as follows: (2) When the ratio [A−]/[HA] = 100:1, HA = H + + A − [A − ] pH = pK a + log The equilibrium constant for this dissociation is [HA ] pH = pK a + log 100 / 1= pK a + 2 [H + ][A − ] Ka = [HA ] (3) When the ratio [A−]/[HA] = 1:10, Cross-multiplication gives pH = pK a + log 1/ 10 = pK a + ( −1) [H+ ][A − ] = K a[HA] If the equation is evaluated at ratios of [A−]/[HA] ranging from 103 to 10−3 and the calculated pH values Divide both sides by [A−]: are plotted, the resulting graph describes the titration curve for a weak acid (Figure 2–4). [HA ] [H + ] = K a [A − ] Solutions of Weak Acids & Their Salts Take the log of both sides: Buffer Changes in pH  [HA ]  Solutions of weak acids or bases and their conjugates log [H + ] = log  K a  exhibit buffering, the ability to resist a change in pH  [A − ]  following addition of strong acid or base. Since many [HA ] metabolic reactions are accompanied by the release or = log K a + log uptake of protons, most intracellular reactions are [A − ] buffered. Oxidative metabolism produces CO2, the an- hydride of carbonic acid, which if not buffered would Multiply through by −1: produce severe acidosis. Maintenance of a constant pH involves buffering by phosphate, bicarbonate, and pro- [HA] − log [H+ ] = − log K a − log teins, which accept or release protons to resist a change [A − ] ch02.qxd 2/13/2003 1:41 PM Page 12 12 / CHAPTER 2 to the pKa. A solution of a weak acid and its conjugate meq of alkali added per meq of acid 1.0 1.0 base buffers most effectively in the pH range pKa ± 1.0 0.8 0.8 pH unit. Figure 2–4 also illustrates the net charge on one Net charge 0.6 0.6 molecule of the acid as a function of pH. A fractional charge of −0.5 does not mean that an individual mole- 0.4 0.4 cule bears a fractional charge, but the probability that a given molecule has a unit negative charge is 0.5. Con- 0.2 0.2 sideration of the net charge on macromolecules as a function of pH provides the basis for separatory tech- 0 0 niques such as ion exchange chromatography and elec- 2 3 4 5 6 7 8 trophoresis. pH Figure 2–4. Titration curve for an acid of the type Acid Strength Depends on HA. The heavy dot in the center of the curve indicates Molecular Structure the pKa 5.0. Many acids of biologic interest possess more than one dissociating group. The presence of adjacent negative charge hinders the release of a proton from a nearby in pH. For experiments using tissue extracts or en- group, raising its pKa. This is apparent from the pKa zymes, constant pH is maintained by the addition of values for the three dissociating groups of phosphoric buffers such as MES ([2-N-morpholino]ethanesulfonic acid and citric acid (Table 2–2). The effect of adjacent acid, pKa 6.1), inorganic orthophosphate (pKa2 7.2), charge decreases with distance. The second pKa for suc- HEPES (N-hydroxyethylpiperazine-N9-2-ethanesulfonic cinic acid, which has two methylene groups between its acid, pKa 6.8), or Tris (tris[hydroxymethyl] amino- carboxyl groups, is 5.6, whereas the second pKa for glu- methane, pKa 8.3). The value of pKa relative to the de- sired pH is the major determinant of which buffer is se- lected. Buffering can be observed by using a pH meter while titrating a weak acid or base (Figure 2–4). We Table 2–2. Relative strengths of selected acids of can also calculate the pH shift that accompanies addi- biologic significance. Tabulated values are the pKa tion of acid or base to a buffered solution. In the exam- values (−log of the dissociation constant) of ple, the buffered solution (a weak acid, pKa = 5.0, and selected monoprotic, diprotic, and triprotic acids. its conjugate base) is initially at one of four pH values. We will calculate the pH shift that results when 0.1 Monoprotic Acids meq of KOH is added to 1 meq of each solution: Formic pK 3.75 Lactic pK 3.86 Acetic pK 4.76 Initial pH 5.00 5.37 5.60 5.86 Ammonium ion pK 9.25 [A−]initial 0.50 0.70 0.80 0.88 [HA]initial 0.50 0.30 0.20 0.12 Diprotic Acids ([A−]/[HA])initial 1.00 2.33 4.00 7.33 Carbonic pK1 6.37 Addition of 0.1 meq of KOH produces pK2 10.25 [A−]final 0.60 0.80 0.90 0.98 Succinic pK1 4.21