Harper-Illustrated-Biochemistry-32nd-Ed.pdf

 a LANGE medical book Harper’s Illustrated Biochemistry THIRTY-SECOND EDITION Peter J. Kennelly, PhD Victor W. Rodwell, PhD Professor Professor (Emeritus) of Bio...

 a LANGE medical book Harper’s Illustrated Biochemistry THIRTY-SECOND EDITION Peter J. Kennelly, PhD Victor W. Rodwell, PhD Professor Professor (Emeritus) of Biochemistry Department of Biochemistry Purdue University Virginia Tech West Lafayette, Indiana Blacksburg, Virginia P. Anthony Weil, PhD Kathleen M. Botham, PhD, DSc Professor Emeritus of Molecular Physiology & Biophysics Emeritus Professor of Biochemistry Vanderbilt University Department of Comparative Biomedical Sciences Nashville, Tennessee Royal Veterinary College University of London London, United Kingdom Owen P. McGuinness, PhD Professor Department of Molecular Physiology & Biophysics Vanderbilt University School of Medicine Nashville, Tennessee New York Chicago San Francisco Athens London Madrid Mexico City Milan New Delhi Singapore Sydney Toronto Copyright © 2023 by McGraw Hill, LLC. All rights reserved. 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Under no circumstances shall McGraw-Hill Education and/or its licensors be liable for any indirect, incidental, special, punitive, consequential or similar damages that result from the use of or inability to use the work, even if any of them has been advised of the possibility of such damages. This limitation of liability shall apply to any claim or cause whatsoever whether such claim or cause arises in contract, tort or otherwise. CoAuthors David A. Bender, PhD Robert K. Murray, MD, PhD Proessor (Emeritus) o Nutritional Biochemistry Emeritus Proessor o Biochemistry University College London University o oronto London, United Kingdom oronto, Ontario, Canada Peter L. Gross, MD, MSc, FRCP(C) Margaret L. Rand, PhD Associate Proessor Senior Associate Scientist Department o Medicine Division o Hematology/Oncology McMaster University Hospital or Sick Children, oronto Hamilton, Ontario, Canada Proessor, Department o Biochemistry University o oronto, oronto, Canada January D. Haile, PhD Associate Proessor o Biochemistry and Molecular Biology Joe Varghese, MD, PhD Centre College Proessor and Head o Biochemistry Danville, Kentucky Christian Medical College Vellore, amil Nadu, India Molly Jacob, MD, PhD, MNASc, FRCPath Proessor o Biochemistry Christian Medical College Vellore, amil Nadu, India Peter A. Mayes, PhD, DSc Emeritus Proessor o Veterinary Biochemistry Royal Veterinary College University o London London, United Kingdom iii This page intentionally left blank Contents Preace ix S E C T I O N 10 The Biochemical Roles o Transition Structures & Functions of Metals 96 I Proteins & Enzymes 1 Peter J. Kennelly, PhD 1 Biochemistry & Medicine 1 S E C T I O N Victor W. Rodwell, PhD Bioenergetics 109 2 Water & pH 6 Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD III 11 Bioenergetics: The Role o ATP 109 3 Amino Acids & Peptides 15 Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD 12 Biologic Oxidation 115 4 Proteins: Determination o Primary Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc Structure 24 Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD 13 The Respiratory Chain & Oxidative Phosphorylation 121 5 Proteins: Higher Orders o Structure 34 Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD S E C T I O N S E C T I O N Metabolism of Enzymes: Kinetics, II Mechanism, Regulation, & Role of Transition Metals 49 IV Carbohydrates 133 14 Overview o Metabolism & the Provision o 6 Proteins: Myoglobin & Hemoglobin 49 Metabolic Fuels 133 Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD Owen P. McGuinness, PhD 7 Enzymes: Mechanism o Action 59 15 Saccharides (ie, Carbohydrates) o Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD Physiological Signicance 147 Owen P. McGuinness, PhD 8 Enzymes: Kinetics 71 Victor W. Rodwell, PhD 16 The Citric Acid Cycle: A Pathway Central to Carbohydrate, Lipid, & Amino Acid 9 Enzymes: Regulation o Activities 85 Metabolism 156 Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD Owen P. McGuinness, PhD v vi CONTENTS 17 Glycolysis & the Oxidation o Pyruvate 163 28 Catabolism o Proteins & o Amino Acid Owen P. McGuinness, PhD Nitrogen 279 Victor W. Rodwell, PhD 18 Metabolism o Glycogen 171 Owen P. McGuinness, PhD 29 Catabolism o the Carbon Skeletons o Amino Acids 290 19 Gluconeogenesis & the Control o Victor W. Rodwell, PhD Blood Glucose 180 Owen P. McGuinness, PhD 30 Conversion o Amino Acids to Specialized Products 306 20 The Pentose Phosphate Pathway & Other Victor W. Rodwell, PhD Pathways o Hexose Metabolism 191 Owen P. McGuinness, PhD 31 Porphyrins & Bile Pigments 315 Victor W. Rodwell, PhD S E C T I O N Metabolism of Lipids 205 V S E C T I O N Structure, Function, & Replication of Informational 21 Lipids o Physiologic Signicance 205 Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc VII Macromolecules 329 32 Nucleotides 329 22 Oxidation o Fatty Acids: Ketogenesis 217 Victor W. Rodwell, PhD Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 33 Metabolism o Purine & Pyrimidine 23 Biosynthesis o Fatty Acids & Nucleotides 337 Eicosanoids 226 Victor W. Rodwell, PhD Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 34 Nucleic Acid Structure & Function 348 24 Metabolism o Acylglycerols & P. Anthony Weil, PhD Sphingolipids 239 Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 35 DNA Organization, Replication, & Repair 360 25 Lipid Transport & Storage 247 P. Anthony Weil, PhD Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 36 RNA Synthesis, Processing, & 26 Cholesterol Synthesis, Transport, & Modication 384 Excretion 259 P. Anthony Weil, PhD Kathleen M. Botham, PhD, DSc, & Peter A. Mayes, PhD, DSc 37 Protein Synthesis & the Genetic Code 404 P. Anthony Weil, PhD S E C T I O N Metabolism of Proteins & VI Amino Acids 273 38 Regulation o Gene Expression 420 P. Anthony Weil, PhD 27 Biosynthesis o the Nutritionally 39 Molecular Genetics, Recombinant DNA, & Nonessential Amino Acids 273 Genomic Technology 444 Victor W. Rodwell, PhD P. Anthony Weil, PhD  CONTENTS vii S E C T I O N S E C T I O N Biochemistry of Extracellular & Intracellular Special Topics (B) 581 VIII Communication 467 X 49 Intracellular Trafc & Sorting o Proteins 581 40 Membranes: Structure & Kathleen M. Botham, PhD, DSc, & Robert K. Murray, MD, PhD Function 467 P. Anthony Weil, PhD 50 The Extracellular Matrix 599 Kathleen M. Botham, PhD, DSc, & Robert K. Murray, MD, PhD 41 The Diversity o the Endocrine System 488 P. Anthony Weil, PhD 51 Muscle & the Cytoskeleton 618 January D. Haile, PhD, & Peter J. Kennelly, PhD 42 Hormone Action & Signal 52 Plasma Proteins & Immunoglobulins 634 Transduction 508 Peter J. Kennelly, PhD P. Anthony Weil, PhD 53 Red Blood Cells 653 Peter J. Kennelly, PhD S E C T I O N 54 White Blood Cells 664 Special Topics (A) 527 IX Peter J. Kennelly, PhD 43 Nutrition, Digestion, & S E C T I O N Absorption 527 Special Topics (C) 677 David A. Bender, PhD XI 44 Micronutrients: Vitamins & Minerals 535 55 Hemostasis & Thrombosis 677 Peter L. Gross, MD, MSc, FRCP(C), David A. Bender, PhD P. Anthony Weil, PhD, & Margaret L. Rand, PhD 45 Free Radicals & Antioxidant 56 Cancer: An Overview 689 Nutrients 549 Molly Jacob, MD, PhD, MNASc, FRCPath, David A. Bender, PhD Joe Varghese, MD, PhD, & P. Anthony Weil, PhD 46 Glycoproteins 554 57 The Biochemistry o Aging 717 David A. Bender, PhD Peter J. Kennelly, PhD 47 Metabolism o Xenobiotics 564 58 Biochemical Case Histories 729 David A. Bender, PhD & Robert K. Murray, MD, PhD David A. Bender, PhD 48 Clinical Biochemistry 568 The Answer Bank 741 David A. Bender, PhD Index 745 This page intentionally left blank Preace Te authors and publishers are pleased to present the thirty- For example, in Chapter 6 the description o the Bohr eect’s second edition o Harper’s Illustrated Biochemistry. Te rst contributions to CO2 transport and release rom the lungs has edition, entitled Harper’s Biochemistry, was published in been reorganized and expanded, while Chapter 9 has been 1939 under the sole authorship o Dr Harold Harper at the updated and reorganized to include expanded coverage o University o Caliornia School o Medicine, San Francisco, zymogen activation in enzyme regulation. Caliornia. Presently entitled Harper’s Illustrated Biochemistry, the book continues, as originally intended, to provide a con- Organization of the Book cise survey o aspects o biochemistry most relevant to the study o medicine. Various authors have contributed to sub- All 58 chapters o the thirty-second edition place major emphasis sequent editions o this medically oriented biochemistry text, on the medical relevance o biochemistry. opics are organized which is now observing its 83rd year. under 11 major headings. In order to assist study and to acilitate retention o the contained inormation, Questions ollow each Section. An Answer Bank ollows Chapter 58. Cover Illustration for Section I includes a brie history o biochemistry and the Thirty-Second Edition emphasizes the interrelationships between biochemistry Te global COVID-19 pandemic has provided a dramatic, and medicine. Water and the importance o homeostasis ace-to-ace demonstration o both the power and limita- o intracellular pH are reviewed, and the various orders tions o molecular medicine and epidemiology. Te rapid o proteins structure are addressed. development o highly eective vaccines was made possible Section II begins with a chapter on hemoglobin. Te by the adaptation o novel RNA-based approaches in which next our chapters address the mechanism o action, the patient’s immune response is activated via the endogenous kinetics, metabolic regulation o enzymes, and the expression o genetically-encoded antigens, rather than the role o metal ions in multiple aspects o intermediary physical injection o a non-inectious antigen. Utilizing the metabolism. patient’s own cells as the bioreactor or generating antigens, Section III addresses bioenergetics and the role o rather than some animal or culture, enabled scientists to use high-energy phosphates in energy capture and transer, the sel-ampliying capacity o polynucleotides to accelerate the oxidation–reduction reactions involved in biologic both the speed o vaccine development and subsequent large- oxidation, and metabolic details o energy capture via scale manuacture. Te illustration on the cover o the thirty- the respiratory chain and oxidative phosphorylation. second edition depicts a neutralizing antibody, in blue, bound Section IV considers the metabolism o carbohydrates to the spike protein on the surace o the SARS-CoV-2 coro- via glycolysis, the citric acid cycle, the pentose phosphate navirus, better known as COVID-19, which is shown in red. pathway, glycogen metabolism, gluconeogenesis, and the Te epitope to which the antibody binds overlaps that at which control o blood glucose. the virus binds to the ACE-2 receptor, the membrane protein by which the pathogen recognizes, binds to, and subsequently Section V outlines the nature o simple and complex invades human cells. Terapeutic antibodies thus protect by lipids, lipid transport and storage, the biosynthesis and physically blocking association o the Spike protein with the degradation o atty acids and more complex lipids, and ACE-2 receptor. the reactions and metabolic regulation o cholesterol biosynthesis and transport in human subjects. Section VI discusses protein catabolism, urea Changes in the Thirty-Second Edition biosynthesis, and the catabolism o amino acids, and As always, Harper’s Illustrated Biochemistry continues to stresses the medically signicant metabolic disorders emphasize the close relationship o biochemistry to the under- associated with their incomplete catabolism. Te nal standing o diseases, their pathology, and the practice o medi- chapter in this section considers the biochemistry o the cine. With the retirement o long-time contributor David A. porphyrins and bile pigments. Bender, Pro. Owen P. McGuinness o Vanderbilt University Section VII rst outlines the structure and unction o has joined as a new coauthor. In addition to the resh per- nucleotides and nucleic acids, and then details DNA spectives and novel insights provided by Pro. McGuinness, replication and repair, RNA synthesis and modication, the contents o most chapters have been updated and provide protein synthesis, the principles o recombinant DNA the reader with the most current and pertinent inormation. technology, and the regulation o gene expression. ix x PREFACE Section VIII considers aspects o extracellular and Acknowledgments intracellular communication. Specic topics include Te authors thank Michael Weitz or his role in the planning membrane structure and unction, the molecular bases o this edition and Peter Boyle or overseeing its preparation o the actions o hormones, and signal transduction. or publication. We also thank asneem Kauser and her col- Sections IX, X, and XI address many topics o leagues at KnowledgeWorks Global Ltd. or their eorts in signicant medical importance. managing editing, typesetting, and artwork. We grateully Section IX discusses nutrition, digestion, and absorption, acknowledge numerous suggestions and corrections received micronutrients including, vitamins, ree radicals rom students and colleagues rom around the world. and antioxidants, glycoproteins, the metabolism o xenobiotics, and clinical biochemistry. Peter J. Kennelly Section X addresses intracellular trafc and the sorting Kathleen M. Botham o proteins, the extracellular matrix, muscle and the Owen P. McGuinness cytoskeleton, plasma proteins and immunoglobulins, Victor W. Rodwell and the biochemistry o red cells and o white cells. P. Anthony Weil Section XI includes hemostasis and thrombosis, an overview o cancer, the biochemistry o aging, and a selection o case histories.  S E C T I O N Structures & Functions I of Proteins & Enzymes C H A P T E R Biochemistry & Medicine Victor W. Rodwell, PhD 1 OBJ E C TI VE S Understand the importance of the ability of cell-free extracts of yeast to ferment sugars, an observation that enabled discovery of the intermediates of After studying this chapter, fermentation, glycolysis, and other metabolic pathways. you should be able to: Appreciate the scope of biochemistry and its central role in the life sciences, and that biochemistry and medicine are intimately related disciplines. Appreciate that biochemistry integrates knowledge of the chemical processes in living cells with strategies to maintain health, understand disease, identify potential therapies, and enhance our understanding of the origins of life on earth. Describe how genetic approaches have been critical for elucidating many areas of biochemistry, and how the Human Genome Project has furthered advances in numerous aspects of biology and medicine. BIOMEDICAL IMPORTANCE DISCOVERY THAT A CELL-FREE Biochemistry and medicine enjoy a mutually cooperative EXTRACT OF YEAST CAN relationship. Biochemical studies have illuminated many FERMENT SUGAR aspects o health and disease, and the study o various aspects o health and disease has opened up new areas o biochem- Although the ability o yeast to “erment” various sugars istry. he medical relevance o biochemistry both in normal to ethyl alcohol has been known or millennia, only com- and abnormal situations is emphasized throughout this book. paratively recently did this process initiate the science o Biochemistry makes signiicant contributions to the ields o biochemistry. he great French microbiologist Louis Pasteur cell biology, physiology, immunology, microbiology, pharma- maintained that ermentation could only occur in intact cells. cology, toxicology, and epidemiology, as well as the ields o However, in 1899, the brothers Büchner discovered that er- inlammation, cell injury, and cancer. hese close relationships mentation could occur in the absence o intact cells when they emphasize that lie, as we know it, depends on biochemical stored a yeast extract in a crock o concentrated sugar solu- reactions and processes. tion, added as a preservative. Overnight, the contents o the crock ermented, spilled over the laboratory bench and loor, 1 2 SECTION I Structures & Functions of Proteins & Enzymes and dramatically demonstrated that ermentation can proceed the interrelationship o biochemistry and medicine is a wide, in the absence o an intact cell. his discovery unleashed an two-way street. Biochemical studies have illuminated many avalanche o research that initiated the science o biochemis- aspects o health and disease, and conversely, the study o vari- try. Investigations revealed the vital roles o inorganic phos- ous aspects o health and disease has opened up new areas o phate, ADP, AP, and NAD(H), and ultimately identiied biochemistry (Figure 1–1). An early example o how investiga- the phosphorylated sugars and the chemical reactions and tion o protein structure and unction revealed the single di- enzymes that convert glucose to pyruvate (glycolysis) or to erence in amino acid sequence between normal hemoglobin ethanol and CO2 (ermentation). Research beginning in the and sickle cell hemoglobin. Subsequent analysis o numerous 1930s identiied the intermediates o the citric acid cycle and variant sickle cell and other hemoglobins has contributed sig- o urea biosynthesis, and revealed the essential roles o certain niicantly to our understanding o the structure and unction vitamin-derived coactors or “coenzymes” such as thiamin both o hemoglobin and o other proteins. During the early pyrophosphate, ribolavin, and ultimately coenzyme A, coen- 1900s, the English physician Archibald Garrod studied patients zyme Q, and cobamide coenzyme. he 1950s revealed how with the relatively rare disorders o alkaptonuria, albinism, cys- complex carbohydrates are synthesized rom, and broken tinuria, and pentosuria, and established that these conditions down into simple sugars, and the pathways or biosynthesis were genetically determined. Garrod designated these condi- o pentoses, and the catabolism o amino acids and atty acids. tions as inborn errors of metabolism. His insights provided Investigators employed animal models, perused intact a oundation or the development o the ield o human bio- organs, tissue slices, cell homogenates and their subractions, chemical genetics. A more recent example was investigation and subsequently puriied enzymes. Advances were enhanced o the genetic and molecular basis o amilial hypercholester- by the development o analytical ultracentriugation, paper olemia, a disease that results in early-onset atherosclerosis. In and other orms o chromatography, and the post-World addition to clariying dierent genetic mutations responsible War II availability o radioisotopes, principally 14C, 3H, and 32P, or this disease, this provided a deeper understanding o cell as “tracers” to identiy the intermediates in complex pathways receptors and mechanisms o uptake, not only o cholesterol such as that o cholesterol biosynthesis. X-ray crystallogra- but also o how other molecules cross cell membranes. Stud- phy was then used to solve the three-dimensional structures ies o oncogenes and tumor suppressor genes in cancer cells o numerous proteins, polynucleotides, enzymes, and viruses. have directed attention to the molecular mechanisms involved Genetic advances that ollowed the realization that DNA was a in the control o normal cell growth. hese examples illustrate double helix include the polymerase chain reaction, and trans- how the study o disease can open up areas o basic biochemi- genic animals or those with gene knockouts. he methods used cal research. Science provides physicians and other workers to prepare, analyze, puriy, and identiy metabolites and the in health care and biology with a oundation that impacts activities o natural and recombinant enzymes and their three- practice, stimulates curiosity, and promotes the adoption o dimensional structures are discussed in the ollowing chapters. scientiic approaches or continued learning. BIOCHEMICAL PROCESSES BIOCHEMISTRY & MEDICINE UNDERLIE HUMAN HEALTH HAVE PROVIDED MUTUAL ADVANCES Biochemical Research Impacts he two major concerns or workers in the health sciences— Nutrition & Preventive Medicine and particularly physicians—are the understanding and he World Health Organization (WHO) deines health as a state maintenance o health and eective treatment o disease. Bio- o “complete physical, mental, and social well-being and not chemistry impacts both o these undamental concerns, and merely the absence o disease and inirmity.” From a biochemical Biochemistry Nucleic acids Proteins Lipids Carbohydrates Genetic Sickle cell Athero- Diabetes diseases anemia sclerosis mellitus Medicine FIGURE 1–1 A two-way street connects biochemistry and medicine. Knowledge of the biochemical topics listed above the green line of the diagram has clarified our understanding of the diseases shown below the green line. Conversely, analyses of the diseases 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.  CHAPTER 1 Biochemistry & Medicine 3 viewpoint, health may be considered that situation in which all announcement that over 90% o the genome had been sequenced. o the many thousands o intra- and extracellular reactions that his eort was headed by the International Human Genome occur in the body are proceeding at rates commensurate with Sequencing Consortium and by Celera Genomics. Except or the organism’s survival under pressure rom both internal and a ew gaps, the sequence o the entire human genome was external challenges. he maintenance o health requires optimal completed in 2003, just 50 years ater the description o the dietary intake o vitamins, certain amino acids and fatty acids, double-helical nature o DNA by Watson and Crick. he various minerals, and water. Understanding nutrition depends implications or biochemistry, medicine, and indeed or all to a great extent on knowledge o biochemistry, and the sciences o biology, are virtually unlimited. For example, the ability o biochemistry and nutrition share a ocus on these chemicals. to isolate and sequence a gene and to investigate its structure Recent increasing emphasis on systematic attempts to maintain and unction by sequencing and “gene knockout” experi- health and orestall disease, or preventive medicine, includes ments have revealed previously unknown genes and their nutritional approaches to the prevention o diseases such as products, and new insights have been gained concerning atherosclerosis and cancer. human evolution and procedures or identiying disease- related genes. Most Diseases Have a Biochemical Basis Major advances in biochemistry and understanding Apart rom inectious organisms and environmental pollut- human health and disease continue to be made by mutation ants, many diseases are maniestations o abnormalities in genes, o the genomes o model organisms such as yeast, the ruit proteins, chemical reactions, or biochemical processes, each ly Drosophila melanogaster, the roundworm Caenorhabditis o which can adversely aect one or more critical biochemical elegans, and the zebra ish; all organisms that can be geneti- unctions. Examples o disturbances in human biochemistry cally manipulated to provide insight into the unctions o responsible or diseases or other debilitating conditions include individual genes. hese advances can potentially provide electrolyte imbalance, deective nutrient ingestion or absorp- clues to curing human diseases such as cancer and Alzheimer tion, hormonal imbalances, toxic chemicals or biologic agents, disease. Figure 1–2 highlights areas that have developed or and DNA-based genetic disorders. o address these challenges, accelerated as a direct result o progress made in the Human biochemical research continues to be interwoven with studies in Genome Project (HGP). New “-omics” ields ocus on com- disciplines such as genetics, cell biology, immunology, nutrition, prehensive study o the structures and unctions o the mol- pathology, and pharmacology. In addition, many biochemists are ecules with which each is concerned. he products o genes vitally interested in contributing to solutions to key issues such (RNA molecules and proteins) are being studied using the as the ultimate survival o mankind, and educating the public to techniques o transcriptomics and proteomics. A spectacu- support use o the scientiic method in solving environmental lar example o the speed o progress in transcriptomics is the and other major problems that conront our civilization. explosion o knowledge about small RNA molecules as regu- lators o gene activity. Other -omics ields include glycomics, lipidomics, metabolomics, nutrigenomics, and pharma- Impact of the Human Genome Project cogenomics. o keep pace with the inormation generated, on Biochemistry, Biology, & Medicine bioinformatics has received much attention. Other related Initially unanticipated rapid progress in the late 1990s in ields to which the impetus rom the HGP has carried over are sequencing the human genome led in the mid-2000s to the biotechnology, bioengineering, biophysics, and bioethics. Transcriptomics Proteomics Glycomics Lipidomics Metabolomics Nutrigenomics Pharmacogenomics Bioinformatics HGP (Genomics) Bioengineering Biotechnology Biophysics Bioethics Stem cell biology Gene therapy Nanotechnology Molecular diagnostics Systems biology Synthetic biology FIGURE 1–2 The Human Genome Project (HGP) has influenced many disciplines and areas of research. Biochemistry is not listed since it predates commencement of the HGP, but disciplines such as bioinformatics, genomics, glycomics, lipidomics, metabolomics, molecular diagnostics, proteomics, and transcriptomics are nevertheless active areas of biochemical research. 4 SECTION I Structures & Functions of Proteins & Enzymes Deinitions o these -omics ields and other terms appear in Bioinformatics: Te discipline concerned with the collection, the Glossary o this chapter. Nanotechnology is an active area, storage, and analysis o biologic data, or example, DNA, RNA, which, or example, may provide novel methods o diagnosis and protein sequences. and treatment or cancer and other disorders. Stem cell biol- Biophysics: Te application o physics and its techniques to biology and medicine. ogy is at the center o much current research. Gene therapy Biotechnology: Te eld in which biochemical, engineering, and has yet to deliver the promise that it appears to oer, but it other approaches are combined to develop biologic products o seems probable that ultimately will occur. Many new molecu- use in medicine and industry. lar diagnostic tests have developed in areas such as genetic, Gene Terapy: Applies to the use o genetically engineered genes to microbiologic, and immunologic testing and diagnosis. treat various diseases. Systems biology is also burgeoning. he outcomes o research Genomics: Te genome is the complete set o genes o an organism, in the various areas mentioned above will impact tremen- and genomics is the in-depth study o the structures and dously the uture o biology, medicine, and the health sciences. unctions o genomes. Synthetic biology oers the potential or creating living organ- Glycomics: Te glycome is the total complement o simple and isms, initially small bacteria, rom genetic material in vitro complex carbohydrates in an organism. Glycomics is the that might carry out speciic tasks such as cleansing petroleum systematic study o the structures and unctions o glycomes such as the human glycome. spills. All o the above make the 21st century an exhilarating Lipidomics: Te lipidome is the complete complement o lipids time to be directly involved in biology and medicine. ound in an organism. Lipidomics is the in-depth study o the structures and unctions o all members o the lipidome and their interactions, in both health and disease. SUMMARY Metabolomics: Te metabolome is the complete complement o Biochemistry is the science concerned with the molecules metabolites (small molecules involved in metabolism) present present in living organisms, individual chemical reactions and in an organism. Metabolomics is the in-depth study o their their enzyme catalysts, and the expression and regulation o structures, unctions, and changes in various metabolic states. each metabolic process. Biochemistry has become the basic Molecular Diagnostics: Reers to the use o molecular approaches such language o all biologic sciences. as DNA probes to assist in the diagnosis o various biochemical, Despite the ocus on human biochemistry in this text, genetic, immunologic, microbiologic, and other medical conditions. biochemistry concerns the entire spectrum o lie orms, rom Nanotechnology: Te development and application to medicine viruses, bacteria, and plants to complex eukaryotes such as and to other areas o devices such as nanoshells, which are only a human beings. ew nanometers in size (10–9 m = 1 nm). Nutrigenomics: Te systematic study o the eects o nutrients on Biochemistry, medicine, and other health care disciplines genetic expression and o the eects o genetic variations on the are intimately related. Health in all species depends on a metabolism o nutrients. harmonious balance o the biochemical reactions occurring in Pharmacogenomics: Te use o genomic inormation and the body, while disease reects abnormalities in biomolecules, technologies to optimize the discovery and development o new biochemical reactions, or biochemical processes. drugs and drug targets. Advances in biochemical knowledge have illuminated many Proteomics: Te proteome is the complete complement o proteins areas o medicine, and the study o diseases has ofen revealed o an organism. Proteomics is the systematic study o the previously unsuspected aspects o biochemistry. structures and unctions o proteomes and their variations in Biochemical approaches are ofen undamental in illuminating health and disease. the causes o diseases and in designing appropriate therapy. Stem Cell Biology: Stem cells are undierentiated cells that have the Biochemical laboratory tests also represent an integral potential to sel-renew and to dierentiate into any o the adult component o diagnosis and monitoring o treatment. cells o an organism. Stem cell biology concerns the biology o A sound knowledge o biochemistry and o other related basic stem cells and their potential or treating various diseases. disciplines is essential or the rational practice o medicine and Synthetic Biology: Te eld that combines biomolecular techniques related health sciences. with engineering approaches to build new biologic unctions and systems. Results o the HGP and o research in related areas will have Systems Biology: Te eld concerns complex biologic systems a proound inuence on the uture o biology, medicine, and studied as integrated entities. other health sciences. ranscriptomics: Te comprehensive study o the transcriptome, Genomic research on model organisms such as yeast, the ruit the complete set o RNA transcripts produced by the genome y D. melanogaster, the roundworm C. elegans, and the zebra during a xed period o time. sh provides insight into understanding human diseases. APPENDIX GLOSSARY Shown are selected examples o databases that assemble, annotate, Bioengineering: Te application o engineering to biology and and analyze data o biomedical importance. medicine. ENCODE: ENCyclopedia Of DNA Elements. A collaborative eort Bioethics: Te area o ethics that is concerned with the application that combines laboratory and computational approaches to o moral and ethical principles to biology and medicine. identiy every unctional element in the human genome.  CHAPTER 1 Biochemistry & Medicine 5 GenBank: Protein sequence database o the National Institutes o ISDB: International Sequence DataBase that incorporates DNA Health (NIH) stores all known biologic nucleotide sequences and databases o Japan and o the European Molecular Biology their translations in a searchable orm. Laboratory (EMBL). HapMap: Haplotype Map, an international eort to identiy single PDB: Protein DataBase. Tree-dimensional structures o proteins, nucleotide polymorphisms (SNPs) associated with common polynucleotides, and other macromolecules, including proteins human diseases and dierential responses to pharmaceuticals. bound to substrates, inhibitors, or other proteins.  C H A P T E R Water & pH Peter J. Kennelly, PhD, & Victor W. Rodwell, PhD 2 OBJ E C TI VE S Describe the properties of water that account for its surface tension, viscosity, liquid state at ambient temperature, and solvent power. After studying this chapter, Represent the structures of organic compounds that can serve as hydrogen you should be able to: bond donors or acceptors. Explain the role played by entropy in the association and orientation, in an aqueous environment, of hydrophobic and amphipathic molecules. Indicate the quantitative contributions of salt bridges, hydrophobic interactions, and van der Waals forces to stabilizing the 3-D conformation of macromolecules. Explain the relationship of pH to acidity, alkalinity, and the quantitative determinants that characterize weak and strong acids. Calculate the shift in pH that accompanies the addition of a given quantity of acid or base to a buffered solution. Describe what buffers do, how they do it, and the conditions under which a buffer is most effective under physiologic or other conditions. Use the Henderson-Hasselbalch equation to calculate the net charge on a polyelectrolyte at a given pH. BIOMEDICAL IMPORTANCE the pH of extracellular fluid between 7.35 and 7.45. Suspected disturbances of acid-base balance are verified by measuring Water is the predominant chemical component of living the pH of arterial blood and the CO2 content of venous blood. organisms. Its unique physical properties, which include Causes of acidosis (blood pH 7.45) may follow vomiting molecules, derive from water’s dipolar structure and excep- of acidic gastric contents. tional capacity for forming hydrogen bonds. The manner in which water interacts with a solvated biomolecule influences the structure both of the biomolecule and of water itself. An WATER IS AN IDEAL BIOLOGIC excellent nucleophile, water is a reactant or product in many metabolic reactions. Regulation of water balance depends on SOLVENT hypothalamic mechanisms that control thirst, on antidiuretic hormone (ADH), on retention or excretion of water by the Water Molecules Form Dipoles kidneys, and on evaporative loss. Nephrogenic diabetes insipi- A water molecule is an irregular, slightly skewed tetrahe- dus, which involves the inability to concentrate urine or adjust dron with oxygen at its center (Figure 2–1). The corners are to subtle changes in extracellular fluid osmolarity, results from occupied by the two hydrogens and the unshared electrons the unresponsiveness of renal tubular osmoreceptors to ADH. of the remaining two sp3-hybridized orbitals of oxygen. The Water has a slight propensity to dissociate into hydroxide 105° angle between the two hydrogen atoms differs slightly ions and protons. The concentration of protons, or acidity, of from the ideal tetrahedral angle, 109.5°. The strongly electro- aqueous solutions is generally reported using the logarithmic negative oxygen atom in a water molecule attracts electrons pH scale. Bicarbonate and other buffers normally maintain away from the hydrogen nuclei, leaving them with a partial 6  CHAPTER 2 Water & pH 7 H CH3 CH2 O H O 2e H 2e H H CH3 CH2 O H O 105° CH2 CH3 H FIGURE 2–1 The water molecule has tetrahedral geometry. R R II C O H N positive charge, while its two unshared electron pairs consti- RI R III tute a region of local negative charge. This asymmetric charge distribution is referred to as a dipole. FIGURE 2–3 Additional polar groups participate in hydrogen bonding. Shown are hydrogen bonds formed between alcohol and Water’s strong dipole is responsible for its high dielectric water, between two molecules of ethanol, and between the peptide constant. As described quantitatively by Coulomb’s law, the carbonyl oxygen and the peptide nitrogen hydrogen of an adjacent strength of interaction F between oppositely charged particles amino acid. is inversely proportionate to the dielectric constant ε of the surrounding medium. The dielectric constant for a vacuum can participate in hydrogen bonding. The oxygen atoms of is essentially unity; for hexane it is 1.9; for ethanol, 24.3; and aldehydes, ketones, and amides, for example, provide lone for water at 25°C, 78.5. When dissolved in water, the force pairs of electrons that can serve as hydrogen acceptors. Alco- of attraction between charged and polar species is greatly hols, carboxylic acids, and amines can serve both as hydrogen decreased relative to solvents with lower dielectric constants. acceptors and as donors of unshielded hydrogen atoms for Its strong dipole and high dielectric constant enable water to formation of hydrogen bonds (Figure 2–3). dissolve large quantities of charged compounds such as salts. Water Molecules Form Hydrogen Bonds INTERACTION WITH WATER A partially unshielded hydrogen nucleus covalently bound to INFLUENCES THE STRUCTURE OF an electron-withdrawing oxygen or nitrogen atom can interact BIOMOLECULES with an unshared electron pair on another oxygen or nitrogen atom to form a hydrogen bond. Since water molecules con- Covalent & Noncovalent Bonds tain both of these features, hydrogen bonding favors the self- Stabilize Biologic Molecules association of water molecules into ordered arrays (Figure 2–2). The covalent bond is the strongest force that holds mol- On average, each molecule in liquid water associates through ecules together (Table 2–1). Noncovalent forces, while of hydrogen bonds with 3.5 others. These bonds are both rela- lesser magnitude, predominate in stabilizing the folding of tively weak and transient, with a half-life of a few picoseconds. the polypeptides and other macromolecules into the complex Rupture of a hydrogen bond in liquid water requires only about three-dimensional conformations essential to their functional 4.5 kcal/mol, less than 5% of the energy required to rupture a competence (see Chapter 5) as well as the association of bio- covalent O—H bond. The exceptional capacity of this relatively molecules into multicomponent complexes. Examples of the small, 18 g/mol, molecule to form hydrogen bonds profoundly latter include the coalescence of the polypeptide subunits that influences the physical properties of water and accounts for its form the hemoglobin tetramer (see Chapter 6); the association high viscosity, surface tension, and boiling point. Hydrogen bonding enables water to dissolve many TABLE 2−1 Bond Energies for Atoms of Biologic organic biomolecules that contain functional groups which Significance H H H H Energy Energy O O Bond Type (kcal/mol) Bond Type (kcal/mol) H H H H O O—O 34 —O O— 96 O H O H O H H S—S 51 C—H 99 H O C—N 70 —S C— 108 H S—H 81 O—H 110 FIGURE 2–2 Water molecules self-associate via hydrogen C—C 82 —C C— 147 bonds. Shown are the association of two water molecules (left) and a hydrogen-bonded cluster of four water molecules (right). Notice that C—O 84 —N C— 147 water can serve simultaneously both as a hydrogen donor and as a N—H 94 —O C— 164 hydrogen acceptor. 8 SECTION I Structures & Functions of Proteins & Enzymes of the two polynucleotide strands that comprise a DNA double helix (see Chapter 34); and the coalescence of billions of phos- pholipid, glycosphingolipid, cholesterol, and other molecules into the bilayer that constitutes the foundation of the plasma membrane of an animal cell (see Chapter 40). These forces, which can be either attractive or repulsive, involve interactions both within the biomolecule and, most importantly, between it and the water that forms the principal component of the sur- rounding environment. In Water, Biomolecules Fold to Position Hydrophobic Groups Within Their FIGURE 2–4 Hydrophobic interactions are driven by the surrounding water molecules. Water molecules are represented by Interior one red (oxygen) and two blue (hydrogen) circles. The hydrophobic Most biomolecules are amphipathic; that is, they possess surfaces of solute molecules are colored gray and, where present, hydrophilic ones are colored green. A. When the six hydrophobic regions rich in charged or polar functional groups as well as cubes shown are dispersed in water (left), the surrounding water regions with hydrophobic character. Proteins tend to fold with molecules (red oxygens and blue hydrogens) are forced to engage in the R-groups of amino acids with hydrophobic side chains in entropically unfavorable interactions with all 36 faces of the cubes. the interior. Amino acids with charged or polar amino acid However, when the six hydrophobic cubes aggregate together side chains (eg, arginine, glutamate, serine; see Table 3–1) (right), the number of exposed faces is reduced to 22. The aggregate forms and its stability is maintained, not by some attractive force, but generally are present on the surface in contact with water. A because aggregation reduces the number of water molecules that similar pattern prevails in a phospholipid bilayer where the are unfavorably affected by nearly 40%. B. Amphipathic molecules charged “head groups” of phosphatidylserine or phosphatidyl- associate together for the same reason. However, the structure of the ethanolamine contact water while their hydrophobic fatty acyl resulting complex (eg, micelle or bilayer) is determined by the geom- side chains cluster together, excluding water (see Figure 40–5). etries of the hydrophobic (gray) and hydrophilic (green) regions. This pattern minimizes energetically unfavorable contacts between water and hydrophobic groups. It also maximizes surface area and reduce the number of water molecules whose the opportunities for the formation of energetically favorable motional freedom becomes restricted (Figure 2–4). Similarly, charge-dipole, dipole-dipole, and hydrogen bonding interac- in the aqueous environment of the living cell the hydrophobic tions between polar groups on the biomolecule and water. portions of amphipathic biopolymers tend to be buried inside the structure of the molecule, or within a lipid bilayer, mini- mizing contact with water. Hydrophobic Interactions Hydrophobic interaction refers to the tendency of nonpolar compounds to self-associate in an aqueous environment. This Electrostatic Interactions self-association is driven neither by mutual attraction nor by Electrostatic interactions between oppositely charged groups what are sometimes incorrectly referred to as “hydrophobic within or between biomolecules are termed salt bridges. Salt bonds.” Self-association minimizes the disruption of energeti- bridges are comparable in strength to hydrogen bonds but act cally favorable interactions between and is therefore driven by over larger distances. They therefore often facilitate the binding the surrounding water molecules. of charged molecules and ions to proteins and nucleic acids. While the hydrogen atoms of nonpolar groups such as the methylene groups of hydrocarbons do not form hydrogen van der Waals Forces bonds, they do affect the structure of the water with which van der Waals forces arise from attractions between transient they are in contact. Water molecules adjacent to a hydropho- dipoles generated by the rapid movement of electrons in all bic group are restricted in the number of orientations (degrees neutral atoms. Significantly weaker than hydrogen bonds of freedom) that permit them to participate in the maximum but potentially extremely numerous, van der Waals forces number of energetically favorable hydrogen bonds. Maximal decrease as the sixth power of the distance separating atoms formation of multiple hydrogen bonds, which maximizes (Figure 2–5). Thus, they act over very short distances, typi- enthalpy, can be maintained only by increasing the order of cally 2 to 4 Å. the adjacent water molecules, with an accompanying decrease in entropy. It follows from the second law of thermodynamics that the Multiple Forces Stabilize Biomolecules optimal free energy of a hydrocarbon-water mixture is a func- The DNA double helix illustrates the contribution of multiple tion of both maximal enthalpy (from hydrogen bonding) and forces to the structure of biomolecules. While each individual highest entropy (maximum degrees of freedom). Thus, non- DNA strand is held together by covalent bonds, the two strands polar molecules tend to form droplets that minimize exposed of the helix are held together exclusively by noncovalent  CHAPTER 2 Water & pH 9.50 and oligonucleotides are stable in the aqueous environment of Interaction energy (kcaI mol–1) the cell. This seemingly paradoxical behavior reflects the fact.25 that the thermodynamics that govern the equilibrium point of a reaction do not determine the rate at which it will pro- 0 ceed toward its equilibrium point. In the cell, macromolecular catalysts called enzymes accelerate the rate of hydrolytic and A –0.25 other chemical reactions when needed. Proteases catalyze the hydrolysis of proteins into their component amino acids, –0.50 while nucleases catalyze the hydrolysis of the phosphoester bonds in DNA and RNA. Precise and differential control of 3.0 4.0 5.0 6.0 7.0 8.0 enzyme activity, including the sequestration of enzymes in R (Å) specific organelles, enables cells to determine the physiologic FIGURE 2–5 The strength of van der Waals interactions circumstances under which a given biopolymer will be synthe- varies with the distance, R, between interacting species. The force sized or degraded. of interaction between interacting species increases with decreasing distance between them until they are separated by the van der Waals contact distance (see arrow marked A). Repulsion due to interaction Many Metabolic Reactions Involve between the electron clouds of each atom or molecule then super- Group Transfer venes. While individual van der Waals interactions are extremely weak, their cumulative effect is nevertheless substantial for macro- Many of the enzymic reactions responsible for synthesis and molecules such as DNA and proteins which have many atoms in close breakdown of biomolecules involve the transfer of a chemical contact. group G from a donor D to an acceptor A to form an acceptor group complex, A—G: interactions such as hydrogen bonds between nucleotide bases (Watson-Crick base pairing) and van der Waals interac- D—G + A  A—G + D tions between the stacked purine and pyrimidine bases. The The hydrolysis and phosphorolysis of glycogen, for example, double helix presents the charged phosphate groups and polar involve the transfer of glucosyl groups to water or to ortho- hydroxyl groups from the ribose sugars of the DNA backbone phosphate. Since the equilibrium constants for these hydro- to water while burying the relatively hydrophobic nucleotide lysis reactions strongly favor the formation of split products, bases inside. The extended backbone maximizes the distance it follows that many of the group transfer reactions respon- between negatively charged phosphates, minimizing unfavor- sible for the biosynthesis of macromolecules are, in and of able electrostatic interactions (see Figure 34–2). themselves, thermodynamically unfavored. Enzyme catalysts play a critical role in surmounting these barriers by virtue of their capacity to directly link two normally separate reactions WATER IS AN EXCELLENT together. For example, by linking an energetically unfavorable NUCLEOPHILE group transfer reaction to a thermodynamically favorable one Metabolic reactions often involve the attack by lone pairs of such as the hydrolysis of ATP, a new enzyme-catalyzed reac- electrons residing on electron-rich molecules termed nucleo- tion can be generated. The free energy change of this coupled philes upon electron-poor atoms called electrophiles. Nucleo- reaction will be the sum of the individual values for the two philes and electrophiles do not necessarily possess a formal that were linked, one whose net overall change in free energy negative or positive charge. Water, whose two lone pairs of favors the formation of the covalent bonds required for bio- sp3 electrons bear a partial negative charge (see Figure 2–1), polymer synthesis. is an excellent nucleophile. Other nucleophiles of biologic importance include the oxygen atoms of phosphates, alcohols, Water Molecules Exhibit a Slight but and carboxylic acids; the sulfur of thiols; and the nitrogen atoms of amines and of the imidazole ring of histidine. Com- Important Tendency to Dissociate mon electrophiles include the carbonyl carbons in amides, The ability of water to ionize, while slight, is of central importance esters, aldehydes, and ketones and the phosphorus atoms of for life. Since water can act both as an acid and as a base, its ioniza- phosphoesters. tion may be represented as an intermolecular proton transfer that Nucleophilic attack by water typically results in the cleav- forms a hydronium ion (H3O+) and a hydroxide ion (OH−): age of the amide, glycoside, or ester bonds that hold biopoly- H2 O + H2 O  H3O + OH− mers together. This process is termed hydrolysis. Conversely, when monomer units such as amino acids or monosaccha- The transferred proton is actually associated with a cluster of rides are joined or condensed together to form biopolymers, water molecules. Protons exist in solution not only as H3O+ such as proteins or starch, water is a product. but also as multimers such as H5O2+ and H7O3+. The proton is Hydrolysis typically is a thermodynamically favored reac- nevertheless routinely represented as H+, even though it is in tion. Yet, the amide and phosphoester bonds of polypeptides fact highly hydrated. 10 SECTION I Structures & Functions of Proteins & Enzymes Since hydronium and hydroxide ions continuously recom- Kw is numerically equal to the product of the molar concentra- bine to form water molecules, an individual hydrogen or oxy- tions of H+ and OH−: gen cannot be stated to be present as an ion or as part of a water molecule. At one instant it is an ion; an instant later it is part K w = [H+ ][OH− ] of a water molecule. Individual ions or molecules are therefore not considered. We refer instead to the probability that at any At 25°C, Kw = (10−7)2, or 10−14 (mol/L)2. At temperatures below instant in time, a given hydrogen will be present as an ion or 25°C, Kw is somewhat less than 10−14, and at temperatures above as part of a water molecule. Since 1 g of water contains 3.35 × 25°C it is somewhat greater than 10−14. Within the stated limi- 1022 molecules, the ionization of water can be described statis- tations of temperature, Kw equals 10−14 (mol/L)2 for all aqueous tically. To state that the probability that a hydrogen exists as an solutions, even solutions containing acids or bases. We can ion is 0.01 means that at any given moment in time, a hydro- therefore use Kw to calculate the pH of any aqueous solution. gen atom has 1 chance in 100 of being an ion and 99 chances out of 100 of being part of a water molecule. The actual prob- ability of a hydrogen atom in pure water existing as a hydrogen pH IS THE NEGATIVE LOG OF THE ion is approximately 1.8 × 10−9. The probability of its being HYDROGEN ION CONCENTRATION part of a water molecule thus is almost unity. Stated another The term pH was introduced in 1909 by Sörensen, who defined way, for every hydrogen ion or hydroxide ion in pure water, it as the negative log of the hydrogen ion concentration: there are 0.56 billion or 0.56 × 109 water molecules. Hydrogen ions and hydroxide ions nevertheless contribute significantly pH = − log[H+ ] to the properties of water. For dissociation of water, This definition, while not rigorous, suffices for most biochem- + [H ][OH ] − ical purposes. To calculate the pH of a solution: K= [H2 O] 1. Calculate the hydrogen ion concentration [H+]. where the brackets represent molar concentrations (strictly 2. Calculate the base 10 logarithm of [H+]. speaking, molar activities) and K is the dissociation constant. 3. pH is the negative of the value found in step 2. Since 1 mole (mol) of water weighs 18 g, 1 liter (L) (1000 g) of water contains 1000 ÷ 18 = 55.56 mol. Pure water thus is For example, for pure water at 25°C, 55.56 molar. Since the probability that a hydrogen in pure water will exist as a hydrogen ion is 1.8 × 10−9, the molar con- pH = − log[H+ ] = − log10−7 = −(−7) = 7.0 centration of H+ ions (or of OH− ions) in pure water is the product of the probability, 1.8 × 10−9, times the molar concen- This value is also known as the power (English), puissant tration of water, 55.56 mol/L. The result is 1.0 × 10−7 mol/L. (French), or potennz (German) of the exponent, hence the use We can now calculate the dissociation constant K for pure of the term “p.” water: Low pH values correspond to high concentrations of H+ and high pH values correspond to low concentrations of H+. [H+ ][OH− ] [10−7 ][10−7 ] Acids are proton donors and bases are proton acceptors. K= = [H2 O] [55.56] Strong acids (eg, HCl, H2SO4) completely dissociate into anions and protons even in strongly acidic solutions (low pH). Weak = 0.018 × 10−14 = 1.8 ×10−16 mol/L acids dissociate only partially in acidic solutions. Similarly, strong The molar concentration of water, 55.56 mol/L, is too great bases (eg, KOH, NaOH), but not weak bases like Ca(OH)2, are to be significantly affected by dissociation. It is therefore con- completely dissociated even at high pH. Many biochemicals are sidered to be essentially constant. The concentration of pure weak acids. Exceptions include phosphorylated intermediates, water may therefore be incorporated into the dissociation con- whose phosphoryl group contains two dissociable protons, the stant K to provide a useful new constant Kw termed the ion first of which is strongly acidic. product for water: The following examples illustrate how to calculate the pH of acidic and basic solutions. [H+ ][OH− ] Example 1: What is the pH of a solution whose hydrogen K= = 1.8 × 10−16 mol/L [H2O] ion concentration is 3.2 × 10−4 mol/L? K w = ( K )[H2O] = [H+ ][OH− ] pH = − log[H+ ] −16 = (1.8 × 10 mol/L)(55.56mol/L) = − log(3.2 × 10−4 ) −14 2 = 1.00 × 10 (mol/L) = − log(3.2) − log(10−4 ) Note that the dimensions of K are moles per liter and those of = − 0.5 + 4.0 Kw are moles2 per liter2. As its name suggests, the ion product = 3.5  CHAPTER 2 Water & pH 11 Example 2: What is the pH of a solution whose hydroxide that present initially in the water. This assumption is valid ion concentration is 4.0 × 10−4 mol/L? We first define a quan- for dilute solutions of strong bases or acids, but not for weak tity pOH that is equal to −log[OH−] and that may be derived bases or acids. Since weak electrolytes dissociate only slightly from the definition of Kw: in solution, we must use the dissociation constant to calcu- late the concentration of [H+] (or [OH−]) produced by a given K w = [H+ ][OH− ] = 10−14 molarity of a weak acid (or base) before calculating total [H+] (or total [OH−]) and subsequently pH. Therefore, log[H+ ] + log[OH− ] = log10−14 Functional Groups That Are Weak Acids Have Great Physiologic Significance or Many biomolecules contain functional groups that are weak pH + pOH = 14 acids or bases. Carboxyl groups, amino groups, and phosphate esters, whose second dissociation falls within the physiologic To solve the problem by this approach: range, are present in proteins and nucleic acids, most coen- [OH− ] = 4.0 × 10−4 zymes, and most intermediary metabolites. Knowledge of the dissociation of weak acids and bases thus is basic to under- pOH = − log[OH− ] standing the influence of intracellular pH on structure and bio- = − log(4.0 × 10−4 ) logic activity. Charge-based separations such as electrophoresis = − log(4.0) − log(10−4 ) and ion exchange chromatography are also best understood in terms of the dissociation behavior of functional groups. = −0.60 + 4.0 When discussing weak acids, we often refer to the proton- = 3.4 ated species (HA or R—SH) as the acid and the unprotonated species (A− or R—S−) as its conjugate base. Similarly, we may Now refer to the deprotonated form as the base (A− or R—COO−) and pH = 14 − pOH = 14 − 3.4 the protonated form as its conjugate acid (HA or R—COOH). = 10.6 We express the relative strengths of weak acids in terms of the dissociation constants of the protonated form. Follow- Examples 1 and 2 illustrate how the logarithmic pH scale facili- ing are the expressions for the dissociation constant (Ka) for a tates recording and comparing hydrogen ion concentrations representative weak acid, R—COOH, as well as the conjugate that differ by orders of magnitude from one another, 0.00032 M acid, R—NH3+, of the weak base R—NH2. (pH 3.5) and 0.000000000025 M (pH 10.6). Example 3: What are the pH values of (a) 2.0 × 10−2 mol/L R—COOH  R—COO− + H+ KOH and of (b) 2.0 × 10−6 mol/L KOH? The OH− arises from [R —COO− ][H+ ] two sources, KOH and water. Since pH is determined by the Ka = [R—COOH] total [H+] (and pOH by the total [OH−]), both sources must be considered. In the first case (a), the contribution of water to R—NH3+  R—NH2 + H+ the total [OH−] is negligible. The same cannot be said for the [R—NH2 ][H+ ] second case (b): Ka = [R—NH3+ ] Concentration (mol/L) Since the numeric values of Ka for weak acids are negative (a) (b) exponential numbers, we express Ka as pKa, where Molarity of KOH 2.0 × 10−2 2.0 × 10−6 pK a = − log K a − −2 −6 [OH ] from KOH 2.0 × 10 2.0 × 10 Note that pKa is related to Ka as pH is to [H+]. The stronger the − −7 [OH ] from water 1.0 × 10 1.0 × 10−7 acid, the lower is its pKa value. Total [OH−] 2.00001 × 10−2 2.1 × 10−6 Representative weak acids (left), their conjugate bases (center), and pKa values (right) include the following: Once a decision has been reached about the significance of R—CH2 —COOH R—CH2 COO− pK a = 4 − 5 the contribution of water, pH may be calculated as shown in Example 3. R—CH2 —NH3+ R—CH2 —NH2 pK a = 9 − 10 The above examples assume that the strong base KOH H2 CO3 HCO3 − pK a = 6.4 is completely dissociated in solution and that the concentra- − −2 tion of OH− ions was thus equal to that due to the KOH plus H2 PO4 HPO4 pK a = 7.2 12 SECTION I Structures & Functions of Proteins & Enzymes pKa is used to express the relative strengths of both weak Cross-multiplication gives acids and weak bases using a single, unified scale. Under this convention, the relative strengths of bases are expressed [H+ ][A− ] = K a [HA] in terms of the pKa of their conjugate acids. For polyprotic Divide both sides by [A−]: compounds containing more than one dissociable proton, a numerical subscript is assigned to each dissociation, numbered [HA] starting from unity in decreasing order of relative acidity. For a [H+ ] = K a [A − ] dissociation of the type R—NH3+ → R—NH 2 + H + Take the log of both sides: the pKa is the pH at which the concentration of the acid  [HA]  log[H+ ] = log  K a −  R—NH3+ equals that of the base R—NH2.  [A ]  From the above equations that relate Ka to [H+] and to the [HA] concentrations of undissociated acid and its conjugate base, when = log K a + log − [A ] [R—COO− ] = [R—COOH] Multiply through by −1: or when [HA] − log[H+ ] = − log K a − log [R—NH2 ] = [R—NH3+ ] [A− ] then Substitute pH and pKa for −log [H+] and −log Ka, respectively; K a = [H ]+ then [HA] Thus, when the associated (protonated) and dissociated pH = pK a − log [A− ] (conjugate base) species are present at equal concentrations, the prevailing hydrogen ion concentration [H+] is numerically Inversion of the last term removes the minus sign and gives equal to the dissociation constant, Ka. If the logarithms of both the Henderson-Hasselbalch equation sides of the above equation are taken and both sides are multi- plied by −1, the expressions would be as follows: [A − ] pH = pK a + log K a = [H+ ] [HA] − log K a = − log[H+ ] The Henderson-Hasselbalch equation has great predictive value in protonic equilibria. For example, Since −log Ka is defined as pKa, and −log [H+] defines pH, the equation may be rewritten as 1. When an acid is exactly half-neutralized, [A−] = [HA]. Under these conditions, pK a = pH [A − ] 1  that is, the pKa of an acid group is the pH at which the pH = pK a + log = pK a + log   = pK a + 0 [HA] 1  protonated and unprotonated species are present at equal concentrations. The pKa for an acid may be determined by Therefore, at half-neutralization, pH = pKa. adding 0.5 equivalent of alkali per equivalent of acid. The resulting pH will equal the pKa of the acid. 2. When the ratio [A−]/[HA] = 100:1, [A − ] The Henderson-Hasselbalch Equation pH = pK a + log [HA] Describes the Behavior of Weak Acids & pH = pK a + log(100/1) = pK a + 2 Buffers The Henderson-Hasselbalch equation is derived below. 3. When the ratio [A−]/[HA] = 1:10, A weak acid, HA, ionizes as follows: pH = pK a + log(1/10) = pK a + (−1) HA  H+ + A− The equilibrium constant for this dissociation is If the equation is evaluated at ratios of [A−]/[HA] ranging from 103 to 10−3 and the calculated pH values are plotted, the [H+ ][A− ] resulting graph describes the titration curve for a weak acid Ka = [HA] (Figure 2–6).  CHAPTER 2 Water & pH 13 1.0 –1.0 Notice that ΔpH, the change in pH per milliequivalent of meq of alkali added per meq of acid OH− added, depends on the initial pH, with highest resistance 0.