《生物化学》课程教学资源（文献资料）Harper’s Illustrated Biochemistry，26th edition，Robert K. Murray Daryl K. Granner Peter A. Mayes Victor W. Rodwell.pdf_P21-P25

WATER&pH一1taken and both sides are multiplied by -l, the expres-Substitute pH and pK, for -log [H+] and -log K, re-sions would be as follows:spectively; then:K, =[Ht][HA]pH=pKg -log -log K, =-log [H][A-]Inversion of the last term removes the minus signSince -log K, is defined as pK, and -log [H] de-and gives the Henderson-Hasselbalch equation:finespH,theequationmaybe rewrittenaspKa =pH[A"]pH=pK +log ![HA]ie, the pK ofan acid group is the pH at which the protonated and unprotonated species are present at equalThe Henderson-Hasselbalch equation has great preconcentrations.ThepK,foran acidmaybedetermineddictive value in protonic equilibria. For example,by adding 0.5 equivalent of alkali per equivalent of(1) When an acid is exactly half-neutralized, [A]acid. The resulting pH will be the pK, of the acid.[HA]. Under these conditions,TheHenderson-HasselbalchEquation[A"]1pH= pKa +logpKa+log=pKa+0DescribestheBehavior91[HA]ofWeakAcids&BuffersThe Henderson-Hasselbalch equation is derived below.Therefore, at half-neutralization, pH= pKA weak acid, HA, ionizes as follows:(2) When the ratio [A]/[HA] - 100:1,HAH++A-[A~]pH=pKa +logTheequilibrium constantfor this dissociation is[HA] pH=pK,+log 100/1=pK,+2K, ={H*A"][HA](3) When the ratio [A-]/[HA] = 1:10,Cross-multiplication givespH=pKa +log 1/10=pKa+(-1)[H* [A"] = K,[{HA]If the equation is evaluated at ratios of [A]/[HA]ranging from 103 to 10-3 and the calculated pH valuesDivide both sides by [A]:are plotted, the resulting graph describes the titrationcurve for a weak acid (Figure 2-4).[H*] =K, [HA][A"]SolutionsofWeakAcids&TheirSaltsTake the log of both sidesBufferChangesinpHSolutions of weak acids or bases and their conjugates[HA] log [Ht] = log exhibit buffering, the ability to resist a change in pHK[A-following addition of strong acid or base.Sincemany[HA]metabolic reactions are accompanied by the release or=logKa +loguptake of protons,most intracellular reactionsare[A"]buffered.Oxidative metabolism produces CO2,the an-hydrideof carbonicacid,whichif notbuffered wouldMultiply through by -1:produce severe acidosis. Maintenance of a constant pHinvolves buffering by phosphate, bicarbonate, and pro[HA]teins, which accept or release protons to resist a change-log [H*]=-log K, -log[A"]

WATER & pH / 11 taken and both sides are multiplied by −1, the expressions would be as follows: Since −log Ka is defined as pKa, and −log [H+] defines pH, the equation may be rewritten as ie, the pKa of an acid group is the pH at which the protonated and unprotonated species are present at equal concentrations. The pKa for an acid may be determined by adding 0.5 equivalent of alkali per equivalent of acid. The resulting pH will be the pKa of the acid. The Henderson-Hasselbalch Equation Describes the Behavior of Weak Acids & Buffers The Henderson-Hasselbalch equation is derived below. A weak acid, HA, ionizes as follows: The equilibrium constant for this dissociation is Cross-multiplication gives Divide both sides by [A− ]: Take the log of both sides: Multiply through by −1: − −− − log [ ] log log [ ] [ ] H HA A a + = K log [ ] log [ ] [ ] log log [ ] [ ] H HA A HA A a a + =       = + K K − − [ ] [ ] [ ] H HA A a + = K − [ ][ ] [ ] H A HA a + = − K Ka H A HA = + [ ][ ] [ ] − HA H A + + − p pH Ka = K K a a H H = = + + [ ] − − log [ ] log Substitute pH and pKa for −log [H+] and −log Ka, respectively; then: Inversion of the last term removes the minus sign and gives the Henderson-Hasselbalch equation: The Henderson-Hasselbalch equation has great predictive value in protonic equilibria. For example, (1) When an acid is exactly half-neutralized, [A− ] = [HA]. Under these conditions, Therefore, at half-neutralization, pH = pKa. (2) When the ratio [A− ]/[HA] = 100:1, (3) When the ratio [A− ]/[HA] = 1:10, If the equation is evaluated at ratios of [A− ]/[HA] ranging from 103 to 10−3 and the calculated pH values are plotted, the resulting graph describes the titration curve for a weak acid (Figure 2–4). Solutions of Weak Acids & Their Salts Buffer Changes in pH Solutions of weak acids or bases and their conjugates exhibit buffering, the ability to resist a change in pH following addition of strong acid or base. Since many metabolic reactions are accompanied by the release or uptake of protons, most intracellular reactions are buffered. Oxidative metabolism produces CO2, the anhydride of carbonic acid, which if not buffered would produce severe acidosis. Maintenance of a constant pH involves buffering by phosphate, bicarbonate, and proteins, which accept or release protons to resist a change pH p p =+ + K K a a log ( 1/10 = 1) − pH p A HA pH p p a a a = + =+ + K K K log [ ] [ ] log 100 /1= − 2 pH p A HA =+ =+ =+ K KK a aa log p p [ ] [ ] log − 1 1 0 pH p A HA = + Ka log [ ] [ ] − pH p HA A = Ka − − log [ ] [ ] ch02.qxd 2/13/2003 1:41 PM Page 11

121CHAPTER2to the pK. A solution of a weak acid and its conjugate111.0base buffers most effectively in the pH range pK, + 1.0pH unit.0.8Figure 2-4 also illustrates the net charge on one/0.60molecule of the acid as a function of pH. A fractionalechargeof-0.5doesnotmean thatanindividual mole-0.40cule bears a fractional charge, but the probability that agiven molecule has a unit negative charge is 0.5. Con-0.20.2sideration of the net charge on macromolecules as ajobewfunction of pH provides thebasis for separatorytech-01oniques such as ion exchange chromatography and elec-2345678trophoresis.pHAcidStrengthDependsonFigure2-4.TitrationcurveforanacidofthetypeMolecularStructureHA.Theheavydot inthecenterofthecurveindicatesthepK,5.0.Manyacids of biologic interestpossessmorethanonedissociating group.The presence of adjacent negativecharge hinders the release of a proton from a nearbygroup,raising its pK.This is apparent from the pKin pH.For experiments using tissue extracts or en-values for the three dissociating groups of phosphoriczymes, constantpH is maintained bytheaddition ofacid and citric acid (Table 2-2). The effect of adjacentbuffers such as MES ([2-N-morpholino]ethanesulfonicchargedecreaseswithdistance.The secondpKforsuc-acid,pK,6.1),inorganic orthophosphate (pK,7.2)HEPES (N-hydroxyethylpiperazine-N-2-ethanesulfoniccinic acid, which has two methylene groups between itscarboxyl groups, is 5.6, whereas the second pK, for glu-acid,pK6.8),or Tris (trishydroxymethyl) amino-methane,pK 8.3).The value of pK,relative to the desired pH is the major determinant of which buffer is se-lectedBuffering can be observed by using a pH meterTable2-2.Relativestrengthsofselectedacidsofwhile titrating a weak acid or base (Figure 2-4). Webiologicsignificance.TabulatedvaluesarethepK,can also calculatethe pH shift that accompanies addi-values (-log of thedissociation constant)oftion of acid or base to a buffered solution. In the exam-ple, the buffered solution (a weak acid, pK =5.0, andselectedmonoprotic,diprotic,andtriproticacidsits conjugatebase)isinitiallyatoneof fourpH valuesWe will calculate the pH shift that results when 0.1MonoproticAcidsmeq of KOH is added to 1 meq of each solution:PK3.75FormicpKLactic3.86pK4.76Acetic5.37Initial pH5.005.605.869.25pKAmmoniumion0.80[A-]initial0.500.700.88DiproticAcids0.500.300.200.12[HAlinia1.002.334.007.33([A-]/[HA])inial6.37CarbonicpK,Addition of 0.1meq of KOH producespK210.254.21SuccinicpK,0.600.800.900.98[A'lainal5.64pK20.400.200.100.02[HAJenalGlutaricpK,4.341.504.0049.09.00([A]/[HA])nnal5.41pK20.951.69log ([A]/[HA])inal0.1760.6025.955.185.606.69FinalpHTriprotic AcidsApH0.180.600.951.692.15PhosphoricpK,6.82pK212.38pK3CitricpK,3.08Notice that the change in pH per milliequivalent of4.74pK2OHadded depends on the initial pH.The solution re-5.40pK3sists changes in pH most effectively at pH values close

12 / CHAPTER 2 0 0.2 0.4 0.6 0.8 1.0 234567 pH 8 0 0.2 0.4 0.6 0.8 1.0 meq of alkali added per meq of acid Net charge Figure 2–4. Titration curve for an acid of the type HA. The heavy dot in the center of the curve indicates the pKa 5.0. Table 2–2. Relative strengths of selected acids of biologic significance. Tabulated values are the pKa values (−log of the dissociation constant) of selected monoprotic, diprotic, and triprotic acids. Monoprotic Acids Formic pK 3.75 Lactic pK 3.86 Acetic pK 4.76 Ammonium ion pK 9.25 Diprotic Acids Carbonic pK1 6.37 pK2 10.25 Succinic pK1 4.21 pK2 5.64 Glutaric pK1 4.34 pK2 5.41 Triprotic Acids Phosphoric pK1 2.15 pK2 6.82 pK3 12.38 Citric pK1 3.08 pK2 4.74 pK3 5.40 Initial pH 5.00 5.37 5.60 5.86 [A− ]initial 0.50 0.70 0.80 0.88 [HA]initial 0.50 0.30 0.20 0.12 ([A− ]/[HA])initial 1.00 2.33 4.00 7.33 Addition of 0.1 meq of KOH produces [A− ]final 0.60 0.80 0.90 0.98 [HA]final 0.40 0.20 0.10 0.02 ([A− ]/[HA])final 1.50 4.00 9.00 49.0 log ([A− ]/[HA])final 0.176 0.602 0.95 1.69 Final pH 5.18 5.60 5.95 6.69 ∆pH 0.18 0.60 0.95 1.69 in pH. For experiments using tissue extracts or enzymes, constant pH is maintained by the addition of buffers such as MES ([2-N-morpholino]ethanesulfonic acid, pKa 6.1), inorganic orthophosphate (pKa2 7.2), HEPES (N-hydroxyethylpiperazine-N9-2-ethanesulfonic acid, pKa 6.8), or Tris (tris[hydroxymethyl] aminomethane, pKa 8.3). The value of pKa relative to the desired pH is the major determinant of which buffer is selected. Buffering can be observed by using a pH meter while titrating a weak acid or base (Figure 2–4). We can also calculate the pH shift that accompanies addition of acid or base to a buffered solution. In the example, the buffered solution (a weak acid, pKa = 5.0, and its conjugate base) is initially at one of four pH values. We will calculate the pH shift that results when 0.1 meq of KOH is added to 1 meq of each solution: Notice that the change in pH per milliequivalent of OH− added depends on the initial pH. The solution resists changes in pH most effectively at pH values close to the pKa. A solution of a weak acid and its conjugate base buffers most effectively in the pH range pKa ± 1.0 pH unit. Figure 2–4 also illustrates the net charge on one molecule of the acid as a function of pH. A fractional charge of −0.5 does not mean that an individual molecule bears a fractional charge, but the probability that a given molecule has a unit negative charge is 0.5. Consideration of the net charge on macromolecules as a function of pH provides the basis for separatory techniques such as ion exchange chromatography and electrophoresis. Acid Strength Depends on Molecular Structure Many acids of biologic interest possess more than one dissociating group. The presence of adjacent negative charge hinders the release of a proton from a nearby group, raising its pKa. This is apparent from the pKa values for the three dissociating groups of phosphoric acid and citric acid (Table 2–2). The effect of adjacent charge decreases with distance. The second pKa for succinic acid, which has two methylene groups between its carboxyl groups, is 5.6, whereas the second pKa for gluch02.qxd 2/13/2003 1:41 PM Page 12

WATER & pH / 13 taric acid, which has one additional methylene group, is 5.4. pKa Values Depend on the Properties of the Medium The pKa of a functional group is also profoundly influenced by the surrounding medium. The medium may either raise or lower the pKa depending on whether the undissociated acid or its conjugate base is the charged species. The effect of dielectric constant on pKa may be observed by adding ethanol to water. The pKa of a carboxylic acid increases, whereas that of an amine decreases because ethanol decreases the ability of water to solvate a charged species. The pKa values of dissociating groups in the interiors of proteins thus are profoundly affected by their local environment, including the presence or absence of water. SUMMARY • Water forms hydrogen-bonded clusters with itself and with other proton donors or acceptors. Hydrogen bonds account for the surface tension, viscosity, liquid state at room temperature, and solvent power of water. • Compounds that contain O, N, or S can serve as hydrogen bond donors or acceptors. • Macromolecules exchange internal surface hydrogen bonds for hydrogen bonds to water. Entropic forces dictate that macromolecules expose polar regions to an aqueous interface and bury nonpolar regions. • Salt bonds, hydrophobic interactions, and van der Waals forces participate in maintaining molecular structure. • pH is the negative log of [H+]. A low pH characterizes an acidic solution, and a high pH denotes a basic solution. • The strength of weak acids is expressed by pKa, the negative log of the acid dissociation constant. Strong acids have low pKa values and weak acids have high pKa values. • Buffers resist a change in pH when protons are produced or consumed. Maximum buffering capacity occurs ± 1 pH unit on either side of pKa. Physiologic buffers include bicarbonate, orthophosphate, and proteins. REFERENCES Segel IM: Biochemical Calculations. Wiley, 1968. Wiggins PM: Role of water in some biological processes. Microbiol Rev 1990;54:432. ch02.qxd 2/13/2003 1:41 PM Page 13

SECTIONIStructures&Functionsof Proteins&Enzymes3AminoAcids&PeptidesVictorW.Rodwell,PhD,&PeterJ.Kennelly,PhDmore than 20 amino acids, its redundancy limits theBIOMEDICALIMPORTANCEavailable codons tothe 20 L-α-amino acids listed inIn addition to providing the monomer units from whichTable3-1,classified according to the polarityoftheirRthe long polypeptide chains of proteins are synthesized,groups.Both one-and three-letter abbreviations for eachthe L-Ot-amino acids and their derivatives participate inamino acid can be used to represent the amino acids incellular functions as diverse as nerve transmission andpeptides (Table 3-1). Some proteins contain additionalthe biosynthesis of porphyrins, purines, pyrimidinesaminoacidsthatarisebymodificationofanaminoacidand urea.Short polymers of amino acids called peptidesalready present in a peptide. Examples include conver-perform prominentrolesin the neuroendocrinesystemsion of peptidyl proline and lysine to 4-hydroxyprolineas hormones,hormone-releasingfactors,neuromodula-and 5-hydroxylysine; the conversion of peptidyl gluta-tors, or neurotransmitters.Whileproteins contain onlymate to -carboxyglutamate;and the methylation,L-αl-amino acids,microorganisms elaborate peptidesformylation,acetylation,prenylation,and phosphoryla-that contain both D- and L-α-amino acids. Several oftion of certain aminoacyl residues.Thesemodificationsthese peptides are of therapeutic value, including the an-extend the biologic diversity of proteins by altering theirtibiotics bacitracin and gramicidin A and the antitumorsolubility,stability,and interaction with other proteins.agent bleomycin. Certain other microbial peptides aretoxic.The cyanobacterial peptides microcystin andOnly L-α-AminoAcidsOccurinProteinsnodularin are lethal in large doses, while small quantitiespromote the formation of hepatic tumors.Neither hu-With the sole exception of glycine, the α-carbon ofmans nor any otherhigher animals can synthesize 10 ofamino acids is chiral. Although some protein aminothe20 common L-αt-amino acids in amounts adequateacidsaredextrorotatoryandsomelevorotatory,all shareto support infantgrowthortomaintainhealthinadultsthe absolute configuration of L-glyceraldehyde and thusConsequently,the human diet must contain adequateare L-α-amino acids, Several free L-α-amino acids fulfillquantities ofthese nutritionally essential amino acids.important roles in metabolic processes.Examples in-clude ornithine, citrulline, and argininosuccinate thatPROPERTIESOFAMINOACIDSparticipate in urea synthesis; tyrosine in formation ofthyroid hormones; and glutamate in neurotransmitterTheGeneticCodeSpecifiesbiosynthesis.D-Amino acids that occur naturally in-20L-α-AminoAcidsclude free D-serine and D-aspartate in brain tissue,Oftheover300naturallyoccurringaminoacids,20con-D-alanine and D-glutamate in the cell walls of gram-stitute the monomer units of proteins.While a nonre-positive bacteria, and D-amino acids in some nonmam-dundant three-letter genetic code could accommodatemalian peptides and certain antibiotics.14

Amino Acids & Peptides 3 14 Victor W. Rodwell, PhD, & Peter J. Kennelly, PhD SECTION I Structures & Functions of Proteins & Enzymes BIOMEDICAL IMPORTANCE In addition to providing the monomer units from which the long polypeptide chains of proteins are synthesized, the L-α-amino acids and their derivatives participate in cellular functions as diverse as nerve transmission and the biosynthesis of porphyrins, purines, pyrimidines, and urea. Short polymers of amino acids called peptides perform prominent roles in the neuroendocrine system as hormones, hormone-releasing factors, neuromodulators, or neurotransmitters. While proteins contain only L-α-amino acids, microorganisms elaborate peptides that contain both D- and L-α-amino acids. Several of these peptides are of therapeutic value, including the antibiotics bacitracin and gramicidin A and the antitumor agent bleomycin. Certain other microbial peptides are toxic. The cyanobacterial peptides microcystin and nodularin are lethal in large doses, while small quantities promote the formation of hepatic tumors. Neither humans nor any other higher animals can synthesize 10 of the 20 common L-α-amino acids in amounts adequate to support infant growth or to maintain health in adults. Consequently, the human diet must contain adequate quantities of these nutritionally essential amino acids. PROPERTIES OF AMINO ACIDS The Genetic Code Specifies 20 L-Amino Acids Of the over 300 naturally occurring amino acids, 20 constitute the monomer units of proteins. While a nonredundant three-letter genetic code could accommodate more than 20 amino acids, its redundancy limits the available codons to the 20 L-α-amino acids listed in Table 3–1, classified according to the polarity of their R groups. Both one- and three-letter abbreviations for each amino acid can be used to represent the amino acids in peptides (Table 3–1). Some proteins contain additional amino acids that arise by modification of an amino acid already present in a peptide. Examples include conversion of peptidyl proline and lysine to 4-hydroxyproline and 5-hydroxylysine; the conversion of peptidyl glutamate to γ-carboxyglutamate; and the methylation, formylation, acetylation, prenylation, and phosphorylation of certain aminoacyl residues. These modifications extend the biologic diversity of proteins by altering their solubility, stability, and interaction with other proteins. Only L-Amino Acids Occur in Proteins With the sole exception of glycine, the α-carbon of amino acids is chiral. Although some protein amino acids are dextrorotatory and some levorotatory, all share the absolute configuration of L-glyceraldehyde and thus are L-α-amino acids. Several free L-α-amino acids fulfill important roles in metabolic processes. Examples include ornithine, citrulline, and argininosuccinate that participate in urea synthesis; tyrosine in formation of thyroid hormones; and glutamate in neurotransmitter biosynthesis. D-Amino acids that occur naturally include free D-serine and D-aspartate in brain tissue, D-alanine and D-glutamate in the cell walls of grampositive bacteria, and D-amino acids in some nonmammalian peptides and certain antibiotics. ch03.qxd 2/13/2003 1:35 PM Page 14