Principles of Medical Biochemistry 4th Edition by Gerhard Meisenberg, William Simmons ISBN 0323391915 9780323391917 pdf download
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Gerhard Meisenberg, PhD
Department of Medical Biochemistry
Ross University School of Medicine
Roseau, Commonwealth of Dominica, West Indies
William H. Simmons, PhD
Department of Molecular Pharmacology and Therapeutics
Loyola University School of Medicine
Maywood, Illinois
Part ONE
PRINCIPLES OF MOLECULAR STRUCTURE AND FUNCTION 1
Chapter 1
INTRODUCTION TO BIOMOLECULES 2
Water Is the Solvent of Life 2
Water Contains Hydronium Ions and Hydroxyl Ions 3
Ionizable Groups Are Characterized by Their pK Values 4
The Blood pH Is Tightly Regulated 4
Acidosis and Alkalosis Are Common in Clinical Practice 5
Bonds Are Formed by Reactions between Functional Groups 6
Isomeric Forms Are Common in Biomolecules 7
Properties of Biomolecules Are Determined by Their Noncovalent Interactions 8
Triglycerides Consist of Fatty Acids and Glycerol 10
Monosaccharides Are Polyalcohols with a Keto Group or an Aldehyde Group 10
Monosaccharides Form Ring Structures 11
Complex Carbohydrates Are Formed by Glycosidic Bonds 11
Polypeptides Are Formed from Amino Acids 14
Nucleic Acids Are Formed from Nucleotides 14
Most Biomolecules Are Polymers 15
Summary 16
Chapter 2
INTRODUCTION TO PROTEIN STRUCTURE 18
Amino Acids Are Zwitterions 18
Amino Acid Side Chains Form Many Noncovalent Interactions 19
Peptide Bonds and Disulfide Bonds Form the Primary Structure of Proteins 20
Proteins Can Fold Themselves into Many Shapes 22
α-Helix and β-Pleated Sheet Are the Most Common Secondary Structures in Proteins 22
Globular Proteins Have a Hydrophobic Core 23
Proteins Lose Their Biological Activities When Their Higher-Order Structure Is Destroyed 24
The Solubility of Proteins Depends on pH and Salt Concentration 25
Proteins Absorb Ultraviolet Radiation 26
Proteins Can Be Separated by Their Charge or Their Molecular Weight 26
Abnormal Protein Aggregates Can Cause Disease 27
Neurodegenerative Diseases Are Caused by Protein Aggregates 29
Protein Misfolding Can Be Contagious 30
Summary 31
Chapter 3
OXYGEN-BINDING PROTEINS: HEMOGLOBIN AND MYOGLOBIN 33
The Heme Group Is the Oxygen-Binding Site of Hemoglobin and Myoglobin 33
Myoglobin Is a Tightly Packed Globular Protein 34
Red Blood Cells Are Specialized for Oxygen Transport 34
The Hemoglobins Are Tetrameric Proteins 34
Oxygenated and Deoxygenated Hemoglobin Have Different Quaternary Structures 36
Oxygen Binding to Hemoglobin Is Cooperative 36
2,3-Bisphosphoglycerate Is a Negative Allosteric Effector of Oxygen Binding to Hemoglobin 37
Fetal Hemoglobin Has a Higher Oxygen-Binding Affinity Than Does Adult Hemoglobin 38
The Bohr Effect Facilitates Oxygen Delivery 39
Most Carbon Dioxide Is Transported as Bicarbonate 39
Summary 40
Chapter 4
ENZYMATIC REACTIONS 41
The Equilibrium Constant Describes the Equilibrium of the Reaction 41
The Free Energy Change Is the Driving Force for Chemical Reactions 42
The Standard Free Energy Change Determines the Equilibrium 43
Enzymes Are Both Powerful and Selective 43
The Substrate Must Bind to Its Enzyme before the Reaction Can Proceed 44
Rate Constants Are Useful for Describing Reaction Rates 44
Enzymes Decrease the Free Energy of Activation 45
Many Enzymatic Reactions Can Be Described by Michaelis-Menten Kinetics 46
K m and V max Can Be Determined Graphically 47
Substrate Half-Life Can Be Determined for First-Order but Not Zero-Order Reactions 48
K cat /K m Predicts the Enzyme Activity at Low Substrate Concentration 48
Allosteric Enzymes Do Not Conform to Michaelis-Menten Kinetics 48
Enzyme Activity Depends on Temperature and pH 49
Different Types of Reversible Enzyme Inhibition Can Be Distinguished Kinetically 49
Covalent Modification Can Inhibit Enzymes Irreversibly 51
Enzymes Stabilize the Transition State 51
Chymotrypsin Forms a Transient Covalent Bond During Catalysis 52
Summary 53
Chapter 5
COENZYMES 55
Enzymes Are Classified According to Their Reaction Type 55
Adenosine Triphosphate Has Two Energy-Rich Bonds 56
ATP DONATES Phosphate in Phosphorylation Reactions 58
ATP Hydrolysis Drives Endergonic Reactions 59
Cells Always Try to Maintain a High Energy Charge 59
Dehydrogenase Reactions Require Specialized Coenzymes 59
Coenzyme A Activates Organic Acids 60
S-Adenosyl Methionine Donates Methyl Groups 62
Many Enzymes Require a Metal Ion 62
Summary 63
GENETIC INFORMATION: DNA, RNA, AND PROTEIN SYNTHESIS 65
Chapter 6
DNA, RNA, AND PROTEIN SYNTHESIS 66
All Living Organisms Use DNA as Their Genetic Databank 66
DNA Contains Four Bases 68
DNA Forms a Double Helix 68
DNA Can Be Denatured 69
DNA Is Supercoiled 69
DNA Replication Is Semiconservative 70
DNA Is Synthesized by DNA Polymerases 70
DNA Polymerases Have Exonuclease Activities 71
Unwinding Proteins Present a Single-Stranded Template to the DNA Polymerases 72
One of the New DNA Strands Is Synthesized Discontinuously 74
RNA Plays Key Roles in Gene Expression 76
The σ Subunit Recognizes Promoters 76
DNA Is Faithfully Copied into RNA 77
Some RNAs Are Chemically Modified After Transcription 79
The Genetic Code Defines the Structural Relationship between mRNA and Polypeptide 80
Transfer RNA Is the Adapter Molecule in Protein Synthesis 81
Amino Acids Are Activated by an Ester Bond with the 3′ Terminus of the tRNA 82
Many Transfer RNAs Recognize More Than One Codon 83
Ribosomes Are the Workbenches for Protein Synthesis 83
The Initiation Complex Brings Together Ribosome, Messenger RNA, and Initiator tRNA 85
Polypeptides Grow Stepwise from the Amino Terminus to the Carboxyl Terminus 85
Protein Synthesis Is Energetically Expensive 88
Gene Expression Is Tightly Regulated 88
A Repressor Protein Regulates Transcription of the lac Operon in E. coli 88
Anabolic Operons Are Repressed by the End Product of the Pathway 89
Glucose Regulates the Transcription of Many Catabolic Operons 90
Transcriptional Regulation Depends on DNA-Binding Proteins 91
Summary 92
Chapter 7
THE HUMAN GENOME 94
Chromatin Consists of DNA and Histones 94
The Nucleosome Is the Structural Unit of Chromatin 95
Covalent Histone Modifications Regulate DNA Replication and Transcription 95
DNA Methylation Silences Genes 95
All Eukaryotic Chromosomes Have a Centromere, Telomeres, and Replication Origins 97
Telomerase Is Required (but Not Sufficient) for Immortality 97
Eukaryotic DNA Replication Requires Three DNA Polymerases 99
Most Human DNA Does Not Code for Proteins 100
Gene Families Originate by Gene Duplication 100
The Genome Contains Many Tandem Repeats 101
Some DNA Sequences Are Copies of Functional RNAs 101
Many Repetitive DNA Sequences Are (or Were) Mobile 102
L1 Elements Encode a Reverse Transcriptase 103
ALU Sequences Spread with the Help of L1 Reverse Transcriptase 105
Mobile Elements Are Dangerous 105
Humans Have Approximately 20,000 Genes 105
Transcriptional Initiation Requires General Transcription Factors 106
Genes Are Surrounded by Regulatory Sites 107
Gene Expression Is Regulated by DNA-Binding Proteins 107
Long Noncoding RNAs Play Roles in Gene Expression 108
mRNA Processing Starts During Transcription 109
Translational Initiation Requires Many Initiation Factors 111
mRNA Processing and Translation Are Often Regulated 113
Small RNA Molecules Inhibit Gene Expression 115
Mitochondria Have Their Own DNA 116
Human Genomes Are Very Diverse 117
Human Genomes Have Many Low-Frequency Copy Number
Variations 117
Summary 118
Chapter 8
PROTEIN TARGETING AND PROTEOSTASIS 120
A Signal Sequence Directs Polypeptides to the Endoplasmic Reticulum 120
Glycoproteins Are Processed in the Secretory Pathway 121
The Endocytic Pathway Brings Proteins into the Cell 124
Lysosomes Are Organelles of Intracellular Digestion 126
Autophagy Recycles Cellular Proteins and Organelles 127
Poorly Folded Proteins Are Either Repaired or Destroyed 127
Ubiquitin Marks Proteins for Destruction 127
The Proteostatic System Protects Cells from Abnormal Proteins 129
Summary 131
Chapter 9
INTRODUCTION TO GENETIC DISEASES 132
Four Types of Genetic Disease 132
Mutations Occur in the Germline and in Somatic Cells 133
Mutations Are an Important Cause of Poor Health 134
Small Mutations Lead to Abnormal Proteins 134
Most Mutations Are Caused by Replication Errors 135
Mutations Can Be Induced by Radiation and Chemicals 135
Mismatch Repair Corrects Replication Errors 136
Missing Bases and Abnormal Bases Need to Be Replaced 138
Nucleotide Excision Repair Removes Bulky Lesions 139
Repair of DNA Double-Strand Breaks Is Difficult 139
Hemoglobin Genes Form Two Gene Clusters 141
Many Point Mutations in Hemoglobin Genes Are Known 142
Sickle Cell Disease Is Caused by a Point Mutation in the β-Chain Gene 143
SA Heterozygotes Are Protected from Tropical Malaria 144
α-Thalassemia Is Most Often Caused by Large Deletions 145
Many Different Mutations Can Cause β-Thalassemia 146
Fetal Hemoglobin Protects from the Effects of β-Thalassemia and Sickle Cell Disease 147
Polygenic Diseases Have Multiple Genetic Risk Factors 147
Genetic Risk Factors Are Discovered in Genome-Wide Association Studies 148
Summary 149
Chapter 10
VIRUSES 152
Viruses Can Replicate Only in a Host Cell 152
Bacteriophage T4 Destroys Its Host Cell 153
DNA Viruses Substitute Their Own DNA for the Host Cell DNA 153
λ Phage Can Integrate Its DNA into the Host Cell Chromosome 154
RNA Viruses Require an RNA-Dependent RNA Polymerase 157
Retroviruses Replicate through a DNA Intermediate 157
Plasmids Are Small “Accessory Chromosomes” or “Symbiotic Viruses” of Bacteria 160
Bacteria Can Exchange Genes by Transformation and Transduction 160
Jumping Genes Can Change Their Position in the Genome 162
Summary 164
Chapter 11
DNA TECHNOLOGY 165
Restriction Endonucleases Cut Large DNA Molecules into Smaller Fragments 165
Large Probes Are Used to Detect Copy Number Variations 165
Small Probes Are Used to Detect Point Mutations 166
Southern Blotting Determines the Size of Restriction Fragments 167
DNA Can Be Amplified with the Polymerase Chain Reaction 168
PCR Is Used for Preimplantation Genetic Diagnosis 169
Allelic Heterogeneity Is the Greatest Challenge for Molecular Genetic Diagnosis 170
Normal Polymorphisms Are Used as Genetic Markers 170
Tandem Repeats Are Used for DNA Fingerprinting 171
DNA Microarrays Can Be Used for Genetic Screening 172
DNA Microarrays Are Used for the Study of Gene Expression 174
DNA Is Sequenced by Controlled Chain Termination 174
Massively Parallel Sequencing Permits Cost-Efficient Whole-Genome Genetic Diagnosis 175
Gene Therapy Targets Somatic Cells 177
Viruses Are Used as Vectors for Gene Therapy 177
Retroviruses Can Splice a Transgene into the Cell’s Genome 178
Genome Editing Is Based on the Making and Healing of DNA Double Strand Breaks 179
Designer Nucleases Are Used for Genome Editing 180
Antisense Oligonucleotides Can Block the Expression of Rogue Genes 181
Genes Can Be Altered in Animals 181
Tissue-Specific Gene Expression Can Be Engineered into Animals 183
Human Germline Genome Editing Is Technically Possible 184
Summary 184
Chapter 12
BIOLOGICAL MEMBRANES 188
Membranes Consist of Lipid and Protein 188
Phosphoglycerides Are the Most Abundant Membrane Lipids 188
Most Sphingolipids Are Glycolipids 190
Cholesterol Is the Most Hydrophobic Membrane Lipid 191
Membrane Lipids Form a Bilayer 192
The Lipid Bilayer Is a Two-Dimensional Fluid 192
The Lipid Bilayer Is a Diffusion Barrier 193
Membranes Contain Integral and Peripheral Membrane Proteins 194
Membranes Are Asymmetrical 194
Membranes Are Fragile 196
Membrane Proteins Carry Solutes Across the Lipid Bilayer 196
Transport against an Electrochemical Gradient Requires Metabolic Energy 198
Active Transport Consumes ATP 199
Sodium Cotransport Brings Molecules into the Cell 199
Summary 201
Chapter 13
THE CYTOSKELETON 203
The Erythrocyte Membrane Is Reinforced by a Spectrin Network 203
Keratins Give Strength to Epithelia 204
Actin Filaments Are Formed from Globular Subunits 206
Striated Muscle Contains Thick and Thin Filaments 207
Myosin Is a Two-Headed Molecule with ATPase Activity 207
Muscle Contraction Requires Calcium and ATP 210
The Cytoskeleton of Skeletal Muscle Is Linked to the Extracellular Matrix 211
Microtubules Consist of Tubulin 213
Eukaryotic Cilia and Flagella Contain a 9 + 2 Array of Microtubules 214
Cells Form Specialized Junctions with Other Cells and with the Extracellular Matrix 214
Summary 216
Chapter 14
THE EXTRACELLULAR MATRIX 218
Collagen Is the Most Abundant Protein in the Human Body 218
The Tropocollagen Molecule Forms a Long Triple Helix 220
Collagen Fibrils Are Staggered Arrays of Tropocollagen Molecules 220
Collagen Is Subject to Extensive Posttranslational Processing 220
Collagen Metabolism Is Altered in Aging and Disease 222
Many Genetic Defects of Collagen Structure and Biosynthesis Are Known 223
Elastic Fibers Contain Elastin and Fibrillin 224
The Amorphous Ground Substance Contains Hyaluronic Acid 225
Sulfated Glycosaminoglycans Are Covalently Bound to Core Proteins 226
Cartilage Contains Large Proteoglycan Aggregates 227
Proteoglycans Are Synthesized in the ER and Degraded in Lysosomes 228
Mucopolysaccharidoses Are Caused by Deficiency of Glycosaminoglycan-Degrading Enzymes 229
Bone Consists of Calcium Phosphates in a Collagenous Matrix 230
Basement Membranes Contain Type IV Collagen, Laminin, and Heparan Sulfate Proteoglycans 230
Fibronectin Glues Cells and Collagen Fibers Together 231
Summary 232
Chapter 15
EXTRACELLULAR MESSENGERS 236
Steroid Hormones Are Made from Cholesterol 236
Progestins Are the Biosynthetic Precursors of All Other Steroid Hormones 238
Thyroid Hormones Are Synthesized from Protein-Bound Tyrosine 242
T4 Becomes Activated to T3 in the Target Tissues 243
Both Hypothyroidism and Hyperthyroidism Are Common Disorders 244
Insulin Is Released Together with the C-Peptide 245
Proopiomelanocortin Forms Several Active Products 245
Angiotensin Is Formed from Circulating Angiotensinogen 246
Immunoassays Are Used for Determination of Hormone Levels 247
Catecholamines Are Synthesized from Tyrosine 249
Indoleamines Are Synthesized from Tryptophan 249
Histamine Is Produced by Mast Cells and Basophils 251
Neurotransmitters Are Released at Synapses 251
Acetylcholine Is the Neurotransmitter of the Neuromuscular Junction 252
There Are Many Neurotransmitters 252
Summary 254
Chapter 16
INTRACELLULAR MESSENGERS 256
Receptor-Hormone Interactions Are Noncovalent, Reversible, and Saturable 256
Many Neurotransmitter Receptors Are Ion Channels 257
Steroid and Thyroid Hormones Bind to Transcription Factors 258
Seven-Transmembrane Receptors Are Coupled to G Proteins 258
Adenylate Cyclase Is Regulated by G Proteins 260
Hormones Can Both Activate and Inhibit the cAMP Cascade 261
Cytoplasmic Calcium Is an Important Intracellular Signal 263
Phospholipase C Generates Two Second Messengers 263
Both cAMP and Calcium Regulate Gene Transcription 264
Muscle Contraction and Exocytosis Are Triggered by Calcium 265
Atrial Natriuretic Factor Acts Through a Membrane-Bound Guanylate Cyclase 265
Nitric Oxide Stimulates a Soluble Guanylate Cyclase 267
cGMP Is a Second Messenger in Retinal Rod Cells 268
Receptors for Insulin and Growth Factors Are Tyrosine-Specific Protein Kinases 269
Growth Factors and Insulin Trigger Multiple Signaling Cascades 271
Cytokine Receptors Use the JAK-STAT Pathway 271
Many Receptors Become Desensitized After Overstimulation 273
Summary 273
Chapter 17
PLASMA PROTEINS 275
Plasma Proteins Are Both Synthesized and Destroyed in the Liver 275
Albumin Prevents Edema 275
Albumin Binds Many Small Molecules 277
Some Plasma Proteins Are Specialized Carriers of Small Molecules 277
Deficiency of α1-Antitrypsin Causes Lung Emphysema 278
Levels of Plasma Proteins Are Affected by Many Diseases 279
Blood Components Are Used for Transfusions 280
Blood Clotting Must Be Tightly Controlled 281
Platelets Adhere to Exposed Subendothelial Tissue 281
Insoluble Fibrin Is Formed from Soluble Fibrinogen 283
Thrombin Is Derived from Prothrombin 284
Factor X Can Be Activated by the Extrinsic and Intrinsic Pathways 284
Negative Controls Are Necessary to Prevent Thrombosis 286
Plasmin Degrades the Fibrin Clot 287
Abnormalities of Purine-Metabolizing Enzymes Can Cause Gout 509
Gout Can Be Treated with Drugs 509
Summary 510
Chapter 31
MICRONUTRIENTS 511
Riboflavin Is a Precursor of Flavin Mononucleotide and Flavin Adenine Dinucleotide 511
Niacin Is a Precursor of NAD and NADP 512
Thiamine Deficiency Causes Weakness and Amnesia 514
Vitamin B6 Plays a Key Role in Amino Acid Metabolism 515
Pantothenic Acid Is a Building Block of Coenzyme A 516
Biotin Is a Coenzyme in Carboxylation Reactions 516
Folic Acid Deficiency Causes Megaloblastic Anemia 517
Vitamin B12 Requires Intrinsic Factor for Its Absorption 519
Vitamin C Is a Water-Soluble Antioxidant 521
Retinol, Retinal, and Retinoic Acid Are the Active Forms of Vitamin A 523
Vitamin D Is a Prohormone 525
Vitamin E Prevents Lipid Oxidation 526
Many Vitamins and Phytochemicals Are Antioxidants 527
Vitamin K Is Required for Blood Clotting 528
Zinc Is a Constituent of Many Enzymes 528
Copper Participates in Reactions of Molecular Oxygen 529
Some Trace Elements Serve Very Specific Functions 530
Summary 531
Chapter 32
INTEGRATION OF METABOLISM 533
Insulin Is Released in Response to Elevated Glucose 533
Insulin Stimulates the Utilization of Nutrients 534
Protein Synthesis Is Coordinated by the mTOR Complex 535
Glucagon Maintains the Blood Glucose Level 536
Catecholamines Mediate the Flight-or-Fight Response 536
Glucocorticoids Are Released in Chronic Stress 537
Energy Is Expended Continuously 538
Stored Fat and Glycogen Are Degraded between Meals 539
Adipose Tissue Is the Most Important Energy Depot 540
The Liver Converts Dietary Carbohydrates to Glycogen and Fat after a Meal 541
The Liver Maintains the Blood Glucose Level During Fasting 541
Ketone Bodies Provide Lipid-Based Energy During Fasting 544
Obesity Is Common in All Affluent Countries 544
Appetite Control Is the Most Important Determinant of Obesity 546
Obesity Is Related to Insulin Resistance 547
Diabetes Is Caused by Insulin Deficiency or Insulin Resistance 549
In Diabetes, Metabolism Is Regulated as in Starvation 550
Diabetes Is Diagnosed with Laboratory Tests 551
Diabetes Leads to Late Complications 552
Many Drugs Are Available for Diabetes Treatment 553
Contracting Muscle Has Three Energy Sources 553
Catecholamines Coordinate Metabolism during Exercise 555
Physical Exercise Leads to Adaptive Changes 557
Ethanol Is Metabolized to Acetyl-CoA in the Liver 558
Liver Metabolism Is Deranged by Alcohol 560
Alcohol Abuse Leads to Fatty Liver and Liver Cirrhosis 561
Most “Diseases of Civilization” Are Caused by Aberrant Lifestyles 561
Aging Is the Greatest Challenge for Medical Research 564
Antiaging Treatments Are Being Investigated 565
Summary 566
ANSWERS TO QUESTIONS 569
GLOSSARY 571
CREDITS 590
INDEX 591
EXTRA ONLINE-ONLY CASE STUDIES
The Mafia Boss
Viral Gastroenteritis
Death in Installments
A Mysterious Death
To Treat or Not to Treat?
Yellow Eyes
An Abdominal Emergency Shortness of Breath Itching
Abdominal Pain
Rheumatism
A Bank Manager in Trouble Kidney Problems
Gender Blender
Man Overboard! Spongy Bones
Blisters
The Sunburned Child Too Much Ammonia
ANSWERS TO CASE STUDIES
Chapter 1
INTRODUCTION TO BIOMOLECULES
Chapter 2
INTRODUCTION TO PROTEIN STRUCTURE
Chapter 3
OXYGEN BINDING PROTEINS: HEMOGLOBIN AND MYOGLOBIN
Chapter 4
ENZYMATIC REACTIONS
Chapter 5 COENZYMES
Biochemistry is concerned with the molecular workings of the body, and the first question we must ask is about the molecular composition of the normal human body. Table 1.1 lists the approximate composition of the proverbial 75-kg textbook adult. Next to water, proteins and triglycerides are most abundant. Triglyceride (aka fat) is the major storage form of metabolic energy, found mainly in adipose tissue. Proteins are of more general importance. They form the structural backbone of cells and tissues and are responsible for enzymatic catalysis, membrane transport, and cell motility. Carbohydrates, in the form of glucose and the storage polysaccharide glycogen, are substrates for the generation of metabolic energy. They also are covalently linked components of glycoproteins and glycolipids. Soluble inorganic salts are present in all intracellular and extracellular fluids, and insoluble salts, most of which are related to calcium phosphate, give strength and rigidity to the bones.
This chapter introduces the principles of molecular structure, the types of noncovalent interactions between biomolecules, and the structural features of the major classes of biomolecules.
Charles Darwin speculated that life originated in a warm little pond. Perhaps it really was a big warm ocean, but one thing is certain: We are appallingly watery creatures. Almost two-thirds of the adult human body is water (see Table 1.1). The structure of water is simple, with two hydrogen atoms bonded to an oxygen atom at an angle of 105 degrees:
Unlike charges attract each other. Therefore the hydrogen atoms of a water molecule are attracted by the oxygen atoms of other water molecules, forming hydrogen bonds:
Water is a lopsided molecule, with its binding electron pairs displaced toward the oxygen atom. Thus the oxygen atom has a high electron density, and the hydrogen atoms are electron deficient. The oxygen atom has a partial negative charge (δ–), and the hydrogen atoms have partial positive charges (δ+). Therefore the water molecule forms an electrical dipole:
The hydrogen bonds are weak. Only 29 kJ/mol (7 kcal/ mol)1 are needed to break a hydrogen bond in water, but 450 kJ/mol (110 kcal/mol) are required to break a covalent oxygen-hydrogen bond in the water molecule itself. Breaking the hydrogen bonds requires no more than heating the water to 100 °C. The hydrogen bonds determine the physical properties of water, including its boiling point.
All body fluids contain inorganic cations (positively charged ions) such as sodium and potassium, and anions (negatively charged ions) such as chloride and phosphate. Table 1.2 lists the typical ionic compositions of intracellular (cytoplasmic) and extracellular (interstitial) fluids. Interestingly, the extracellular fluid has an ionic composition similar to seawater. We carry a warm little pond with us, a replica of the environment in which our ancestors originated.
Predictably, the cations are attracted to the oxygen atom of the water molecule, and the anions are attracted to the hydrogen atoms. The ion-dipole interactions thus formed are the forces that keep the components of soluble salts in solution, as in the case of sodium chloride (table salt):
higher concentration of carbonic acid in venous blood. Carbonic acid forms from carbon dioxide and water, either spontaneously or catalyzed by the enzyme carbonic anhydrase:
addition, the CO2 level can be regulated by the lungs and the HCO3– level by the kidneys.
Phosphate groups provide an additional buffer system:
CO2 + H2O G G
Carbonic anhydrase Spontaneous HCO3– + H+
H2CO3
At 37 °C and pH 7.4, there are approximately 800 molecules of dissolved CO2 and 16,000 molecules of HCO3 – for every molecule of H2CO3. The apparent pK for the overall reaction CO2 + H2O → HCO3 –+ H+ is 6.1.
The importance of the equilibrium between H2CO3, HCO3–, and H+ is evident when the pH of the solution is disturbed. When acid (H+) is added while HCO3 – is present, most of the H+ will be absorbed by the HCO3 –, forming first H2CO3 and then CO2. Conversely, when alkali is added (H+ removed) H2CO3 releases H+ to the surrounding water while being regenerated from CO2 and H2O. Thus the presence of the weak acid H2CO3 and the weak base HCO3– stabilizes the pH of the solution.
Substances that stabilize the pH are called buffers. All weak acids and weak bases buffer the pH of the solution at pH values close to the pK values of their ionizable groups. Ionizable groups in proteins participate in the maintenance of a constant pH in cells and body fluids. However, carbonic acid/bicarbonate is the most important physiological buffer system in the body. It is important because CO2 and HCO3– are present in high concentrations in the interstitial and intracellular compartments as well as in the plasma (Fig. 1.1). In
= 6.9
The phosphate buffer is important only in the intracellular compartments, in which both inorganic phosphate and organically bound phosphate are plentiful.
Even small deviations from the normal blood pH lead to severe disturbances. An arterial pH lower than 7.35 is called acidemia, and an arterial pH exceeding 7.45 is called alkalemia. The pathological states leading to these outcomes are called acidosis and alkalosis, respectively.
Respiratory acidosis is caused by the abnormal retention of CO2, and respiratory alkalosis is caused by hyperventilation. For example, a doubling in the rate of alveolar ventilation raises the blood pH from 7.40 to 7.62, and a 50% reduction in alveolar ventilation lowers the blood pH from 7.40 to 7.12 (Fig. 1.2).
Metabolic acidosis can be caused by the overproduction of an organic acid, for example:
Table 1.6
Carboxylic
Methyl ethers, some membrane lipids
Triglycerides, other lipids
Disaccharides, oligosaccharides, and polysaccharides (glycosidic bonds)
Nucleotides; most important:
Many metabolic intermediates, phosphoproteins Phosphodiester
Acetyl-CoA, other “activated” acids
Thioether
ATP, adenosine triphosphate; CoA, coenzyme A. * “Energy-rich” bonds.
lumen of the gastrointestinal tract, where neither adenosine triphosphate (ATP) nor other usable energy sources are available.
Some bonds contain more energy than others. Most ester, ether, acetal, and amide bonds require between 4 and 20 kJ/mol (1 and 5 kcal/mol) for their formation, and the same amount of energy is released during their hydrolysis. Anhydride bonds and thioester bonds, however, have free energy contents greater than 20 kJ/
mol. They are classified, rather arbitrarily, as energy-rich bonds.
The biological properties of molecules are determined not only by their composition but by their geometry. Isomers are chemically different molecules with identical
Noncovalent interactions determine the biological properties of biomolecules:
• Water solubility depends on hydrogen bonds and ion-dipole interactions that the molecules form with water. Charged molecules and those that can form many hydrogen bonds are soluble, and those that have mainly nonpolar bonds, for example between C and H, are insoluble. Molecules that can exist in charged and uncharged states are more soluble in the charged form.
• Higher-order structures of macromolecules, including proteins (see Chapter 2) and nucleic acids (see Chapter 6), are formed by noncovalent interactions between portions of the same molecule. Because noncovalent interactions are weak, many are needed to hold a protein or nucleic acid in its proper shape.
• Binding interactions between molecules are the essence of life. Structural proteins bind each other, substrates bind to enzymes, gene regulators bind to deoxyribonucleic acid (DNA), hormones bind to receptors, and foreign substances bind to antibodies.
After this review of functional groups, bonds, and noncovalent interactions, the structures of the major classes of biomolecules—triglycerides, carbohydrates, proteins, and nucleic acids—can now be introduced. Details about these structures are presented in later chapters.
The triacylglycerols, better known as triglycerides in the medical literature, consist of glycerol and fatty acids. Glycerol is a trivalent alcohol:
Palmitic acid
Palmitic acid can also be written as:
(CH2)14 COOH
Fatty acids consist of a long hydrocarbon chain with a carboxyl group at one end. The typical chain length is between 16 and 20 carbons. For example,
Fatty acids that have only single bonds between carbons are called saturated fatty acids. Those with at least one double bond between carbons are called unsaturated fatty acids. For example, H3C (CH2)5 (CH2)7 COOH CH CH
Palmitoleic acid
Fatty acids have pK values between 4.7 and 5.0; therefore they are mainly in the deprotonated (—COO–) form at pH 7.
In the triglycerides, all three hydroxyl groups of glycerol are esterified with a fatty acid, as shown in Fig. 1.4 The long hydrocarbon chains of the fatty acid residues ensure that triglycerides are insoluble in water. In the body, triglycerides minimize contact with water by forming fat droplets.
Collectively, nonpolar biomolecules are called lipids. The triglycerides (“fat”) are used only as a storage form of metabolic energy, but other lipids serve as structural components of membranes (see Chapter 12) or as hormones (see Chapter 15).
Monosaccharides are the building blocks of all carbohydrates. A monosaccharide consists of a chain of carbons with a hydroxyl group at each carbon except one,
Fig. 1.4 Structure of a triglyceride (fat) molecule. Although the ester bonds can form some hydrogen bonds with water, the long hydrocarbon chains of the fatty acids make fat insoluble.
which forms a carbonyl group. Aldoses have an aldehyde group, and ketoses have a keto group. The length of the carbon chain is variable. For example,
• Triose: three carbons
• Tetrose: four carbons
• Pentose: five carbons
• Hexose: six carbons
• Heptose: seven carbons
d-Glyceraldehyde and dihydroxyacetone are the simplest monosaccharides:
have four different substituents. These four asymmetric carbons can form 16 isomers, but only one of them is d-glucose. By convention, the “d” in d-glyceraldehyde and d-glucose refers to the orientation of substituents at the asymmetrical carbon farthest removed from the carbonyl carbon (C-2 and C-5, respectively).
Monosaccharides that differ in the orientation of substituents around one of their asymmetric carbons are called epimers. In Fig. 1.5, for example, d-mannose is a C-2 epimer of glucose, and d-galactose is a C-4 epimer of glucose. Epimers are diastereomers, not enantiomers. They have different physical and chemical properties.
D-Glyceraldehyde (an aldotriose)
Dihydroxyacetone (a ketotriose)
The most important monosaccharide, however, is the aldohexose d-glucose:
1 2 3 4 5 6
The carbons are numbered, starting with the aldehyde carbon or, for ketoses, the terminal carbon closest to the keto carbon. Carbons 2, 3, 4, and 5 of d-glucose all
Most monosaccharides spontaneously form ring structures in which the aldehyde (or keto) group forms a hemiacetal (or hemiketal) bond with one of the hydroxyl groups. If the ring contains five atoms, it is called a furanose ring; if it contains six atoms, it is called a pyranose ring. The ring structures are written in either the Fischer projection or the Haworth projection, as shown in Fig. 1.6.
In water, only one of 40,000 glucose molecules is in the open-chain form. When the ring forms, carbon 1 of glucose becomes asymmetric. Therefore two isomers, αd-glucose and β-d-glucose, can form. These two isomers are called anomers. In glucose, carbon 1 (the aldehyde carbon) is the anomeric carbon. In the ketoses, the keto carbon (usually carbon 2) is anomeric.
Unlike epimers, which are stable under ordinary conditions, anomers interconvert spontaneously. This process is called mutarotation. It is caused by the occasional opening and reclosure of the ring, as shown in Fig. 1.7. The equilibrium between the α- and β-anomers is reached within several hours in neutral solutions, but mutarotation is greatly accelerated in the presence of acids or bases.
Monosaccharides combine into larger molecules by forming glycosidic bonds: acetal or ketal bonds involving the anomeric carbon of one of the participating
(open-chain form)
(open-chain form)
- D-Glucopyranose (modified Fischer projection)
D-Glucopyranose (Haworth projection)
- D-Fructofuranose (modified Fischer projection)
- D-Fructofuranose (Haworth projection)
Fig. 1.6 Ring structures of the aldohexose d-glucose and the ketohexose d-fructose. The six-member pyranose ring is favored in d-glucose, and the five-member furanose ring is favored in d-fructose.
open-chain form (0.0025%)
- D-Glucopyranose (66%)
Fig. 1.7 Mutarotation of d-glucose. Closure of the ring can occur either in the α- or the β-configuration.
monosaccharides. The anomeric carbon forms the bond in either the α- or the β-configuration. Once the bond is formed, mutarotation is no longer possible. For example, maltose and cellobiose in Fig. 1.8 differ only in the orientation of their 1,4-glycosidic bond, but they have different properties and do not convert spontaneously into one another.
Structures formed from two monosaccharides are called disaccharides. Products with three, four, five, or six monosaccharides are called trisaccharides, tetrasaccharides, pentasaccharides, and hexasaccharides, respectively. Oligosaccharides (from Greek ○λιγ○σ meaning “a few”) contain “a few” monosaccharides, and polysaccharides (from Greek π○λυσ meaning “many”) contain “many” monosaccharides (Fig. 1.9).
Carbohydrates can form glycosidic bonds with noncarbohydrates. Glycoproteins contain carbohydrate covalently bound to amino acid side chains. In glycolipids, carbohydrate is covalently bound to a lipid. If the sugar binds its partner through an oxygen atom, the bond is called O-glycosidic; if the bond is through nitrogen, it is called N-glycosidic.
Monosaccharides, disaccharides, and oligosaccharides (“sugars”) are water soluble because they form multiple hydrogen bonds with water. Many polysaccharides, however, are insoluble because their large size increases the opportunities for intermolecular interactions. Things become insoluble when the molecules interact more strongly with one another than with the surrounding water.
Glucose
CH2OH
Galactose
α(1→4) glycosidic bond
Glucose
CH2OH
Glucose
β(1→4) glycosidic bond
Glucose
Maltose
β(1→4) glycosidic bond
Glucose
Lactose
αβ′(1→2) glycosidic bond
Fig. 1.8 Structures of some common disaccharides. By convention, the nonreducing end of the disaccharide is written on the left side and the reducing end on the right side.
→
Fig. 1.9 Structures of some common polysaccharides. A, Amylose is an unbranched polymer of glucose residues in α(1→4)glycosidic linkage. Together with amylopectin—a branched glucose polymer with a structure resembling glycogen—it forms the starch granules in plants. B, Like amylose, cellulose is an unbranched polymer of glucose residues. As a major cell wall constituent of plants, it is the most abundant biomolecule on earth. The marked difference in the physical and biological properties between the two polysaccharides is caused by the presence in cellulose of β(1→4) glycosidic bonds rather than α(1,4) glycosidic bonds. C, Glycogen is the storage polysaccharide of animals and humans. Like amylose, it contains chains of glucose residues in α(1,4) glycosidic linkage. Unlike amylose, however, the molecule is branched. Some glucose residues in the chain form a third glycosidic bond, using their hydroxyl group at carbon 6.
The carbonyl group of the monosaccharides has reducing properties. The reducing properties are lost when the carbonyl carbon forms a glycosidic bond. Of the disaccharides in Fig. 1.8, only sucrose is not a reducing sugar because both anomeric carbons participate in the glycosidic bond. The other disaccharides have a reducing end and a nonreducing end.
Polypeptides are constructed from 20 different amino acids. All amino acids have a carboxyl group and an amino group, both bound to the same carbon. This carbon, called the α-carbon, also carries a hydrogen atom and a fourth group, the side chain, which differs in the 20 amino acids. The general structure of the amino acids can be depicted as follows,
Nucleic acids contain three kinds of building blocks:
1. A pentose sugar, which is ribose in ribonucleic acid (RNA) and 2-deoxyribose in 2-deoxyribonucleic acid (DNA):
where R (residue) is the variable side chain. The α-carbon is asymmetric, but of the two possible isomers, only the l-amino acids occur in polypeptides.
Dipeptides are formed when the carboxyl group of one amino acid reacts with the amino group of another amino acid. The substituted amide bond thus formed is called the peptide bond:
2. Phosphate, which is bound to hydroxyl groups of the sugar.
3. The bases adenine, guanine, cytosine, and either uracil (in RNA) or thymine (in DNA); cytosine, thymine, and uracil are pyrimidines, containing a single six-member ring; adenine and guanine are purines, containing two condensed rings:
Chains of “a few” amino acids are called oligopeptides, and chains of “many” amino acids are called polypeptides.
A nucleoside is obtained when C-1 of ribose or 2-deoxyribose forms an N-glycosidic bond with one of the bases (Fig. 1.10). Nucleotides consist of a sugar, a base, and up to three phosphate groups bound to C-5 of the sugar. They are named as phosphate derivatives of the nucleosides. Thus adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (ATP) contain one, two, and three phosphates, respectively.
Nucleic acids are polymers of nucleoside monophosphates. The phosphate group forms a phosphodiester bond between the 5′- and 3′-hydroxyl groups of
2. The molecule shown here is acetylsalicylic acid (aspirin). What kind of electrical charge does aspirin carry in the stomach at a pH value of 2 and in the small intestine at a pH value of 7?
A. Negatively charged in the stomach; positively charged in the intestine
B. Negatively charged both in the stomach and the intestine
C. Uncharged in the stomach; negatively charged in the intestine
D. Uncharged both in the stomach and the intestine
E. Uncharged in the stomach; positively charged in the intestine
3. Inorganic phosphate, which is a major anion in the intracellular space, has three acidic functions with pK values of 2.3, 6.9, and 12.3, as shown below. In skeletal muscle fibers, the intracytoplasmic pH is about 7.1 at rest and 6.6 during vigorous anaerobic exercise. What does this mean for inorganic phosphate in muscle tissue?
A. Phosphate molecules absorb protons when the pH decreases during anaerobic exercise.
B. On average, the phosphate molecules carry more negative charges during anaerobic contraction than at rest.
C. Phosphate molecules release protons when the pH decreases during anaerobic exercise.
D. The most abundant form of the phosphate molecule in the resting muscle fiber carries one negative charge.
Each class of biomolecules has its own specialized functions. Nucleic acids are repositories of genetic information and play multiple roles in the expression of this information. Membrane lipids form the backbone of membranes and function as diffusion barriers between the interior of cells and their environment and between the cellular organelles.
Proteins, however, are required for each and every cellular function. Membrane proteins join hands with the fibrous proteins of cytoplasm and extracellular matrix to keep cells and tissues in shape, DNA-binding proteins regulate gene expression, and metabolism depends on enzyme proteins that catalyze chemical reactions and on transporters that bring nutrients and metabolic intermediates across the cellular membranes.
This chapter introduces the 20 amino acids that occur in proteins. It describes the covalent structure of proteins and the ways in which noncovalent forces fold the proteins into the three-dimensional shapes that are required for their biological functions.
All amino acids have an α-carboxyl group, an α-amino group, a hydrogen atom, and a variable side chain R (“residue”), all bound to the α-carbon. This structure forms two optical isomers:
The pK of the α-carboxyl group is around 2.0, and the pK of the α-amino group is near 9 or 10. The protonation state varies with the pH (Fig. 2.1). At a pH below the pK of the carboxyl group, the amino acid is predominantly a cation; above the pK of the amino group, the amino acid is an anion; and between the two pK values, the amino acid is a zwitterion (from German zwitter meaning “hermaphrodite”), that is, a molecule carrying both a positive and a negative charge. The isoelectric point (pI)
3N CH
9.9 H3N CH H2N CH + GG
Fig. 2.1 Protonation states of the amino acid alanine. The zwitterion is the predominant form in the pH range from 2.3 to 9.9. mL NaOH added
Only the l-amino acids occur in proteins. d-Amino acids are rare in nature, although they occur in some bacterial products.
Fig. 2.2 Titration curve of the amino acid alanine. The two level segments are caused by the buffering capacity of the carboxyl group (at pH 2.3) and the amino group (at pH 9.9).
is defined as the pH value at which the number of positive charges equals the number of negative charges. For a simple amino acid such as alanine, the pI is halfway between the pK values of the two ionizable groups. Note that whereas the pK is the property of an individual ionizable group, the pI is a property of the whole molecule.
The pK values of the ionizable groups are revealed by treating an acidic solution of an amino acid with a strong base or by treating an alkaline solution with a strong acid. Any ionizable group stabilizes the pH at values close to its pK because it releases protons when the pH in its environment rises, and it absorbs protons when the pH falls. The titration curve shown in Fig. 2.2 has two flat segments that indicate the pK values of the two ionizable groups.
The titration curves of amino acids that have an additional ionizable group in the side chain show three rather than two buffering areas. The pI of the acidic amino acids is halfway between the pK values of the two acidic groups, and the pI of the basic amino acids is halfway between the pK values of the two basic groups (Fig. 2.3).
The 20 amino acids can be placed in a few major groups (Fig. 2.4). Their side chains form noncovalent interactions in the proteins, and some form covalent bonds:
1. Small amino acids: Glycine and alanine occupy little space. Glycine, in particular, is found in places where two polypeptide chains have to come close together.
2. Branched-chain amino acids: Valine, leucine, and isoleucine have hydrophobic side chains.
Fig. 2.3 Prevailing ionization states of the amino acids aspartate (A) and lysine (B) at different pH values. The isoelectric points of aspartate and lysine are 2.95 and 10.0, respectively.
3. Hydroxyl amino acids: Serine and threonine form hydrogen bonds with their hydroxyl group. They also form covalent bonds with carbohydrates and with phosphate groups.
4. Sulfur amino acids: Cysteine and methionine are quite hydrophobic, although cysteine also has weak acidic properties. The sulfhydryl (—SH) group of cysteine can form a covalent disulfide bond with another cysteine side chain in the protein.
5. Aromatic amino acids: Phenylalanine, tyrosine, and tryptophan are hydrophobic, although the side chains of tyrosine and tryptophan can also form hydrogen bonds. The hydroxyl group of tyrosine can form a covalent bond with a phosphate group.
6. Acidic amino acids: Glutamate and aspartate have a carboxyl group in the side chain that is negatively charged at pH 7. The corresponding carboxamide groups in glutamine and asparagine are not acidic but form strong hydrogen bonds. Asparagine is an attachment point for a carbohydrate in glycoproteins.
7. Basic amino acids: Lysine, arginine, and histidine carry a positive charge on the side chain, although the pK of the histidine side chain is quite low.
8. Proline is a freak among amino acids, with its nitrogen tied into a ring structure as a secondary amino group. Being stiff and angled, it often forms a bend in the polypeptide.
The pK values of the ionizable groups in amino acids and proteins are summarized in Table 2.1. Most negative charges in proteins are contributed by the side chains of glutamate and aspartate, and most positive charges are contributed by the side chains of lysine and arginine.
Fig. 2.5 Structure of polypeptides. Note the polarity of the chain, with a free amino group at one end of the chain and a free carboxyl group at the opposite end.
terminus
Peptide bonds
The peptide bond is conventionally written as a single bond, with four substituents attached to the carbon and nitrogen of the bond:
α-Carbon
Carbonyl carbon
Hydrogen
Nitrogen
Oxygen
Side chain
terminus
A C—N single bond, like a C—C single bond, should show free rotation. The triangular plane formed by the O C—Cα1 portion should be able to rotate out of the plane of the Cα2—N—H portion. Actually, however, the peptide bond is a resonance hybrid of two structures:
of the peptide bond
Its “real” structure is between these two extremes. One consequence is that, like C C double bonds (see Chapters 12 and 25 ), the peptide bond does not rotate. Its four substituents are frozen in space with the two α -carbons in trans configuration, opposite each other.
The other two bonds in the polypeptide backbone, those formed by the α-carbon, are “pure” single bonds with the expected rotational freedom. Rotation around the nitrogen—carbon bond is measured as the φ (phi) angle, and rotation around the peptide bond carbon—α-carbon bond as the ψ (psi) angle (Fig. 2.6). This rotational freedom turns polypeptides into contortionists capable of forming U-turns and spirals.
Globular proteins have compact shapes. Most are water soluble, but some are embedded in cellular membranes or form supramolecular aggregates, such as the ribosomes. Hemoglobin and myoglobin (see Chapter 3), enzymes (see Chapter 4), membrane proteins (see Chapter 12), and plasma proteins (see Chapter 17) are globular proteins. Fibrous proteins are long and
2.6 Geometry of the peptide bond. The φ and ψ angles are variable.
threadlike, and most serve structural functions. The keratins of hair, skin, and fingernails are fibrous proteins (see Chapter 13), as are the collagen and elastin of the extracellular matrix (see Chapter 14).
α-HELIX AND β-PLEATED SHEET ARE THE MOST COMMON SECONDARY STRUCTURES IN PROTEINS
A secondary structure is a regular, repetitive structure that emerges when all the φ angles in the polypeptide are the same and all the ψ angles are the same. Only a few secondary structures are energetically possible.
In the α-helix (Fig. 2.7), the polypeptide backbone forms a right-handed corkscrew. “Right-handed” refers
The noncovalent interactions in proteins are so weak that the higher-order structure of proteins can be destroyed by heating. Within a few minutes of being heated above a certain temperature (often between 50 ° C and 80 ° C), the higher-order structure unravels into a messy tanglework. This process is called heat denaturation.
Denaturation destroys the protein’s biological properties. Stated another way, the biological properties of proteins require intact higher-order structures. Also the physical properties of the protein change dramatically with denaturation. For example, water solubility is lost because the denatured polypeptide chains become irrevocably entangled. Generally, protein denaturation is irreversible. A boiled egg does not become unboiled when it is kept in the cold.
Not only heat, but anything that disrupts noncovalent interactions can denature proteins. Detergents and organic solvents denature proteins by disrupting
hydrophobic interactions. Being nonpolar, they insert themselves between the side chains of hydrophobic amino acids. Strong acids and bases denature proteins by changing their charge pattern. In a strong acid, the protein loses its negative charges; in a strong base, it loses its positive charges. This deprives the protein of intramolecular salt bonds. Also, high concentrations of small hydrophilic molecules with high hydrogen bonding potential, such as urea, can denature proteins. They do so by disrupting the hydrogen bonds between water molecules. This limits the extent to which water molecules are forced into a thermodynamically unfavorable “ordered” position at an aqueous-nonpolar interface, weakening the hydrophobic interactions within the protein.
Heavy metal ions (lead, cadmium, mercury) can denature proteins by binding to carboxylate groups and, in particular, sulfhydryl groups in proteins. Their affinity for functional groups in proteins is one reason for the toxicity of heavy metals.
The fragility of life is appalling. A 6 ° C rise of the body temperature can be fatal, and the blood pH must never fall below 7.0 or rise above 7.7 for any length of time. These subtle changes in the physical environment do not cleave covalent bonds, but they disrupt noncovalent interactions. It is because of the vulnerability of noncovalent higher-order structures that living beings had to evolve homeostatic mechanisms for the maintenance of their internal environments.
Unlike fibrous proteins, most globular proteins are water soluble. Their solubility is affected by the salt concentration. Raising the salt concentration from 0% to 1% or more increases their solubility because the salt ions neutralize the electrical charges on the protein, thereby reducing electrostatic attraction between neighboring protein molecules (Fig. 2.11, A and B). Very high salt concentrations, however, precipitate proteins because most of the water molecules become tied up in the hydration shells of the salt ions. Effectively, the salt competes with the protein for the available solvent.
The addition of a water-miscible organic solvent (e.g., ethanol) can precipitate proteins because the organic solvent competes for the available water. Unlike denaturation, precipitation is reversible and does not permanently destroy the protein’s biological properties.
The pH value is also important. When the pH is at the protein’s pI, the protein carries equal numbers of positive and negative charges. This maximizes the opportunities for the formation of intermolecular salt bonds, which glue the protein molecules together into insoluble aggregates or crystals (Fig. 2.11, A and C). Therefore the solubility of proteins is minimal at their isoelectric point.
Fig. 2.11 Effects of salt and pH on protein solubility. A, Protein in distilled water. Salt bonds between protein molecules cause the molecules to aggregate. The protein becomes insoluble. B, Protein in 5% sodium chloride (NaCl). Salt ions bind to the surface charges of the protein molecules, thereby preventing intermolecular salt bonds. C, The effect of pH on protein solubility. The formation of intermolecular salt bonds is favored at the isoelectric point. At pH values much greater or less than the pI, the electrostatic interactions between the molecules are mainly repulsive.
Proteins do not absorb visible light. Therefore they are uncolored unless they contain a colored prosthetic group, such as the heme group in hemoglobin or the cofactor retinal in the visual pigment rhodopsin. They do, however, absorb ultraviolet radiation with two absorption maxima. One absorbance peak, at 190 nm, is caused by the peptide bonds. A second peak, at 280 nm, is caused by aromatic amino acid side chains. The peak at 280 nm is more useful in laboratory practice because it is relatively specific for proteins. Nucleic acids, however, have an absorbance peak at 260 nm that overlaps the 280-nm peak of proteins (Fig. 2.12).
Dialysis is used in the laboratory to separate proteins from salts and other small contaminants. The protein is enclosed in a little bag of porous cellophane (Fig. 2.13). The pores allow salts and small molecules to diffuse out, but the large proteins are retained. The dialysis of kidney patients is based on the same principle (Clinical Example 2.1).
Electrophoresis separates proteins according to their charge-mass ratio, based on their movement in an electrical field. At pH values above the protein’s pI, the protein carries mainly negative charges and moves to the
Fig. 2.12 Typical ultraviolet absorbance spectra of proteins and nucleic acids. The protein absorbance peak at 280 nm is caused by the aromatic side chains of tyrosine and tryptophan. Nucleic acids absorb at 260 nm because of the aromatic character of their purine and pyrimidine bases.
Fig. 2.13 Use of dialysis for protein purification. Only small molecules and inorganic ions can pass through the porous membrane.
anode; at pH values below the protein’s pI, it carries mainly positive charges and moves to the cathode. At the pI, the net charge is zero, and the protein stays put.
Electrophoresis on cellulose acetate foil, starch gel, and other carrier materials is the standard method for separation of plasma proteins and detection of abnormal proteins in the clinical laboratory (Fig. 2.14, A). When a structurally abnormal protein differs from its normal counterpart by a single amino acid substitution, the electrophoretic mobility is changed only if the charge pattern is changed. For example, when a glutamate residue is replaced by aspartate, the electrophoretic mobility remains the same because these two amino acids carry the same charge. However, when glutamate is replaced by an uncharged amino acid such as valine, one negative charge is removed, and the two proteins can be separated by electrophoresis.
Between 40% and 50% of the blood volume is occupied by blood cells. The remaining fluid, called plasma, is a solution containing about 0.9% inorganic ions, 7% protein, and low concentrations of nutrients including 0.1% glucose. Water-soluble waste products such as urea (containing nitrogen from amino acid breakdown) and uric acid (from purine nucleotides) are present, but their concentrations are low because they are removed continuously by the kidneys. In patients with kidney failure, these waste products accumulate to dangerous levels. The standard treatment for these patients is hemodialysis. In this procedure, the patient’s blood is passed along semipermeable membranes. The pores in these membranes are large enough to allow the passage of low-molecular-weight waste products (but also salts and nutrients), but plasma proteins and blood cells are retained. The blood is dialyzed not against distilled water (which would lead to a malpractice suit) but against a solution with physiological concentrations of nutrients and inorganic ions.
Electrophoresis can be performed in a cross-linked polyacrylamide or agarose gel that impairs the movement of large molecules. At a pH at which all proteins move to the same pole, the molecules are separated mainly by their molecular weight rather than their charge–mass ratio (Fig. 2.14, B).
Ordinarily, proteins that have lost their native conformation are destroyed by proteases, either within or outside the cells. In some cases, however, misfolded proteins arrange into fibrils that are difficult to degrade. A typical pattern is seen in this process. A globular protein that has a somewhat flexible higher-order structure
Fig. 2.14 Protein separation by electrophoresis. A, On a wet cellulose acetate foil, the proteins are separated according to their net charge. If an alkaline pH is used, as in this case, the proteins are negatively charged and move to the anode. B, Electrophoresis in a cross-linked polyacrylamide gel. Although small molecules can move in the field, larger ones “get stuck” in the gel. Under suitable pH conditions, this method separates on the basis of molecular weight rather than charge.
in its normal state spontaneously refolds into a state with a high content of β-pleated sheet. In some cases, stretches of α-helix rearrange into stretches of β-pleated sheet. Unlike the α-helix, which is strictly intramolecular, the β-pleated sheet can form extended structures that involve two or more polypeptides. Therefore these pH = 8.6