BIOCHEMISTRY electrolyte disorders, particularly hypernatremia and hyponatremia (of sodiury); hyp hypokalemia (of potassium) Diuretics in the tre: and hypertension Diuretics are the drugs that and sodium excretion, so that uh increased. The commonly used bendrofluazide, frusemide, spiconol. mannitol. Diuretics are important in the treatment of edema, hear failure and hypertendion, ee ‘The normal pH of the blood is maintained in the narow range of 7.35-7.45, ic. slightly alkaline, The pH of intracellular fluid is rather variable. Thus, for erythrocytes the pH is 7.2, while fr skeletal muscle, it may be as low as 6.0. Maintenance of blood pH is an important homeostatic mechanism of the body. In normal circumstances, the regulation is so effective that the blood pH varies very little. Changes in blood pH will alter the intracellular pH which, in turn, influence the metabolism eg. distortion in protein structure, enzyme activity etc. It is estimated that the blood pH compatible to life is 6.8.7.8. (For a good understanding of acid-base balance, adequate knowledge on acids, bases, pH and buffers is essential (reier Chapter 40, Production of acids by the body| ‘The metabolism of the body is accompanied by an overall production of acids. These include the volatile acids like carbonic acid (most predominent, about 20,000 mEq/day) ot non- volatile acids (about 80 mEo/éay) such as lactic acid, sulfuric acid, phosphoric acid etc. Carbonic acid is formed from the metabolic product COy; lactic acid is produced in anaerobic metabolism; sulfuric acid is generated from proteins (sulfur containing amino acids); phosphoric acid is derived from organic phosphates (e.g. phospholipids). All these acids add up H* ions to the blood. A diet rich in animal proteins results in more acid production by the body that ultimately leads to the excretion of urine which is profoundly acidic. Production of bases by the The formation of basic compounds ihe body, in the normal circumstances, is negligible. Some amount of bicarbonate is generated from the organic acids such as lactate and citrate. A vegetarian diet has a tendency for a net production of bases. This is due to the fact that vegetarian diet produces salts of organic acids such as sodium lactate which can utilize H* ions produced in the body. For this reason, a vegetarian diet has an alkalizing effect on the body. This is reflected by the excretion of neutral or slightly alkaline urine by these subjectsPan ‘The body has developed three lines of defense to regulate the body's acid-base balance and maintain the blood pH (around 7.4). 1. Blood butlers 1. Respiratory mechanism Il, Renal mechanism. 1. Blood buffers A bufier may be defined as a solution of a weak acid (HA) and its salt (BA) with a strong Base. The buler resist the change inp by the addition of acid or alkali and the buffering Capacty is dependent on. the absolute concentration o sat and acid should be bore im mind thatthe buffer cannot remove H* fons from the body. It temporarily acts as a shock absorbant to reduce the fee H* ons, The H* ions have to be ultimately eliminated by the renal mechanism (Gescried later The blood contains 3 buffer systems, 1 Bicarbonate bute 2. Phosphate buffer 3, rotein bter 1. Bicarbonate bufer system + Sodium bi carbonate an eatbonic aid (NaHCO, ~ HCO,) isthe most predominant bur system of the estacelsfr td Gatbonic ac dsociates nto hydrogen and brcarbonate ons. H,CO, =H" + HCO; By the law of mass action, at equim [r*] co] “THoy Dissociation constant of HCO) ‘The equation may be rewritten as follows. por] =x aL * [reo] ‘We know that pH = i 0 2 1 Hy By taking the reciprocals and logarithms (for logs, multiplication becomes addition fxcor | * es cesy (3) The equation 3 may now be written as ico: [c,] The above equation is valid for any buffer pair. The general equation refered to at Hendersontasslbalch equation fr any bullet is writen as 6 It is evident from tis equation that the pH is dependent on ratio of the concentration of the base to acid (HCO and HCO, in equation 4). flood pH and the ratio of HCO; to H,CO, : The plasma bicarbonate (HCO; concentration is around 24 mmol/l (range 22-26 mmolf)). Carbonic acid is a solution of CO in water, Its concentration is given by the product cof pC? (arterial partial pressure of CO, = 40 mm Hg) and the solubility constant of Particularly the plasma. {203 Thus HCO, = 40 x 0.03 = 1.2 mmol, el The Henderson-Hasselbalch equation for bicarbonate buffer is Substituting the values Wlood pH = 7.4; 0 = pk, for H,CO, = 6.1; HCO; = 24 mmol/l; HCO, = 1.2 mmol), in the above equation = 6.1 + log 20 = 61413 74476 BIOCHEMISTRY It is evident that at a blood pH 7.4, the ratio. of bicarbonate to carbonic acid is 20 : 1. Thus, the bicarbonate concentration Is much higher (20 times) than carbonic acid in the blood. This is referred to as alkali, and is responsible {or the effective'Duffering of H* ions, generated in the body. In normal circumstances, the concentration of bicarbonate and carbonic acid determines the pH of blood. Further, the bicarbonate buffer system serves as an index to understand the disturbances in the acid-base balance of the body. 2. Phosphate buffer system = Sodium dihydrogen phosphate and disodium hydrogen phosphate (NaHPO, ~ NajHPO,) constitute the phosphate buffer. It is mostly an intracellular buffer and is of less importance in Blasma due To its low concentration. With a pK of 6.8 (close to blood pH 7.4), the phosphate buffer would have been more effective, had it been present in high concentration. It is estimated that the ratio of base to acid for phosphate butler is 4 compared to 20 for bicarbonate buffer. 3. Protein buffer system : The plasma proteins and hemoglobin together constitute the protein buffer system of the blood. The buffering Capacity of proteins is dependent on the pk of ionizable groups of amino acids. The imidazole group of histidine pk = 6.7) s the most effective contributor of proiein bulfers. The plasma proteins account for about 2% of the total buffering capacity of the plasma. Hemoglobin of RBC is also an important buffer. it mainly buffers the fixed acids, besides being involved in the transport of gases (O, and CO,). More details on hemoglobin are given under respiratory mechanism for regulation of pH. piratory mechanism for pH regulation Respiratory system provides a rapid mechanism for the maintenance of acid-base balance. This is achieved by regulating the Concentration of carbonic acid (HCO3) in the blood ie. the denominator in the bicarbonate buffer system. The details of CO, transport and the role of hemoglobin in this process are described elsewhere (Chapter 10, Refer Fig. 10.6) The large volumes of CO, produced by the cellular metabolic activity endanger the acid- base equilibrium of the body. But in normal incumstances, all of this CO, is eliminated from the body in the expired air via the lungs, as summarized below. Caton nbydese H,co, "+ co, + H,0- The rate of respiration (or the rate of removal ‘of COs) is contiolled by a respiratory centre, located in the medulla of the brain, This centre is highly sensitive to changes in the pH of blood ‘Any decrease in blood pH causes hyperventilation to blow off CO,, thereby reducing the HCO; concentration. Simultaneously, the H* ions are eliminated as H,0. Respiratory control of blood pH is rapid but only a short term regulatory process, since hyperventilation cannot proceed for long. Hemoglobin as a buffer : Hemoglobin of ‘erythrocytes is also important in the respiratory regulation of pH. At the tissue level, hemoglobin binds to H* ions and helps to transport CO, as HCO} with a minimum change in pH (referred to as isohydric transport). In the lungs, as hemoglobin combines with O2, H* ions are removed which combine with HCO} to form HyCO3, The latter dissociates to release CO, to be exhaled! (Refer Fig.10.6). Generation of HCO} by RBC : Due to lack of aerobic metabolic pathways, RBC produce very little CO., The plasma CO, diffuses into the RBC along the concentration gradient where it combines with water to form H,CO3. This reaction is catalysed by carbonic anhydrase (also called carbonate dehydratase). In the RBC, HCO; dissociates to produce H* and HCO} The Ht ions are trapped and buffered by hemoglobin. As the concentration of HCO; increases in the RBC, it diffuses into plasma along with the concentration gradient, in exchange for CI ions, to maintain electrical neutrality. This phenomenon, referred to as chloride shift, helps to generate HCOF (Fig.21.4)Chapter 21: WATER, ELECTROLYTE AND ACIDSASE BALANCE a7z Plasma co, + 00. +H20 The role of kidneys in the maintenance of acid-base balance of the body (blood pH) is highly significant. The renal mechanism tres to provide a permanent solution to the acid-base Seen Tr s celel ocdceanearsy buffering. system and a short term respiratory mechanism, described above ‘The kidneys regulate the blood pH by maintaining the alkali reserve, besides excreting Cr reabsorbing the acidic or basic substances, as the situation demands. Urine pH normally lower than blood pH : The pH of urine is normally acidic This clearly indicates that the kidneys. Mee contributed to the acidification of urine, when it is formed from the blood plasma (pHi 7.4). In other words, the H* jons generated in the body in the normal circumstances, are eliminated by acidified urine. Hence the pH of urine is normally acidic (~6.0), while that of blood is alkaline (7.4). Urine pH, however, is variable ‘and may range between 4.5.9.5, depending on the concentration of Ht ions. Carbonic anhydrase and renal regulation of pH = The enzyme carbonic anhydrase (inhibited by acetazolamide) is of central importance in the renal regulation of pH which occurs by the following mechanisms. retion of H ions /Reabsorption of bicarbonate Cexcretion of titratable acié —sbreton of ammonium ons Excretion of H* ions : Kidney is the only route through which the H* can be eliminated from the body. Ht excretion occurs in the proximal convoluted tubules (renal tubular cells) and is coupled with the regeneration of HCO}. The process depicted in Fig.27.5, occurs as follows. Carbonic anhydrase catalyses the production of carbonic acid (HCO) from CO, and HO in the renal tubular cell. HCO; then dissociates to H* and HCO3, The H* ions are secreted into the tubular lumen in exchange for Nat. The Nat in association with HCO} is reabsorbed into the blood. This is an effective mechanism to eliminate acids (H*) from the body with a simultaneous generation of HCO}. The latter ‘adds up to the alkali reserve of the body. The H* combines with a non-carbonate base and is scree Aateorton of bicarbonate: This mecha- nism is primarily responsible to conserve the blood HCO3, with a simultaneous excretion of H® fons, The normal urine is almost free from HCO3, This is explained as follows (Fig.21.6) Tubuler lumen Na*K are ther beo BIOCHEMISTRY woes [Roar] ‘eee - = eHt HCO3 Ae wi Fitered) HCO HaCO3 tex |e 0+00, +f. 4.0 aR Bicarbonate freely diffuses from the plasma into the tubular lumen, Here HCO} combines with H*, secreted by tubular cells, to form HCOs. HCO} is then cleaved by carbonic anhydrase (of tubular cell membrane) to form CO} and H;0. As the CO» concentration builds up in the lumen, it diffuses into the tubular cells along the concentration gradient. In the tubular cell, CO, again combines with HO to form HCO} which then dissociates into H* and HCO}. The H* is secreted into the lumen in exchange for Na*. The HCO¥ is reabsorbed into plasma in association with Na*. Reabsorption of HCO} is a cyclic process with the net excretion of H* or generation of new HCO}. This is because the H* is derived from water. This mechanism helps to maintain the steady state and will not be effective for the elimination of HY or geggamtion of new HCOs. yom of titratable acid acihty is a measure of acid excreted into urine Titratable by the kidney. This can be estimated by titrating urine back to the normal pH of blood (7.4). In ‘quantitative terms, titratable acidity refers to the number of milliliters of N/10 NaOH required to titrate 1 liter of urine to pH 7.4. Titratable acidity reflects the H* ions excreted into urine which resulted in a fall of pH from 7.4 (that of blood) ‘The excreted H* ions are actually buffered in the turine by phosphate buffer as depicted in Fig.21.7, and briefly described hereunder. As already discussed, H* ion is secreted into the tubular lumen in exchange for Na* ion. T Nat is obtained from the base, disodium hydrogen phosphate (NagHPO,). The latter in tum combines with H* to produce the acid, sodium dihydrogen phosphate (NaHzPOs), in which form the major quantity of titratable acid in urine is present. As the tubular fluid moves down the renal tubules, more and more Hi ions are added, resulting in the acidification of urine. ‘This causes a fall in the pH of urine to as low as 4.5. Any further fall in the pH will cause depletion of Na* ions. yns = This is H* fons secreted H* ion combines with Blood ena tubular cot “Abul men NazHPO, pHT4 Nat Nat Na" Ly NaHPO HCO3. HCO3 +H" Ht. H:co, NaHzPO, I slat ls Renal regulation of blood pH-Excretion of tatabie acid buffer machanism (CA-Carbonic anhyaso)‘ ( Dog ommonia Boffer Gia Clides / Guyton ov : WATER, ELECTROLYTE AND ACID-AASE BALANCE 479 i Renal tubular cet! Gtuanine z Fig. 21.8: Renal regulation of biood pHi-Excrotion of ‘ammonium fons (CA~Carbonic anhydrase) NH, to form ammonium ion (NH?) Ammonium ions cannot diff cells and, therefore, are e fi. eis estimated that of body acid load is of NHf ions. For this fon via NH} excretion is iminate large quantities of the body. This mechanism inant panicularly in acidosis about half to two-thig reason, renal_regul very effective to Carbon dioxide—the central molecule of pH regulation ‘As is observed from the foregoing discussion, CO, is of central importance in the acid-base balance of the body. thas the ability to combine with H0 to from HyCO3 which can dissociate to HCO} and H*. A summary of the interaction between the lungs, erythrocytes and kidneys in handling COz to maintain pH of the blood is depicted in Fig.21.9. The CO; generated by aerobic metabolism may be exhaled via lungs, ‘or converted to HCOs by erythrocytes and kidneys to add up to the alkali reserve of the body. Butfers of intracellular fluids The regulation of pH within the cells is as Important as that discussed above for the extracellular fluid. The H* ions generated in the cells are exchanged for Na* and K* ions. This is particularly observed in skeletal muscle which reduces the potential danger of H* accumulation in the cells. Pend Dae The body has developed an eificient system for the maintenance of acid-base equilibrium with a result that the pH of blood is almost constant (7.4). The blood pH compatible to life is 6.8-7.8, beyond which life cannot exist For a better understanding of the disorders of acid-base balance, the Henderson-Hasselbalch ‘equation must be frequently consulted. ico) pH = pk, + log: [reo] pH74 Metabolism C0, Lungs (60. generated) HC.) | (COsexhales) HCO3; Enthiooytes Kidnoys (C0. tansported, (HC0% gene- HCO; generated) rated," ost Fig. 21.9: Carbon doxide—the contra molecule of ‘boad pH regulation.CHAPTER Water & pH Peter J. Kennelly, PhD & Victor W. Rodwell, PhD CESSES) = Describe the properties of water that account for its surface tension, viscosity, liquid state at ambient temperature, and solvent power, © Use structural formulas to represent several organic compounds that can serve {as hydrogen bond donors or acceptors. = Explain the role played by entropy in the orientation, in an aqueous environment, of the polar and nonpolar regions of macromolecules. = Indicate the quantitative contributions of salt bridges, hydrophobic interactions, and van der Weals forces to the stability 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 the pH of a buffered sol "= Describe what buffers do, how they do it, and the conditions under which a buffer is most effective under physiologic or other conditions. ‘= Illustrate how the Henderson-Hasselbalch equation can be used to calculate the net charge on a polyelectrolyte at a given pH. Ta ee BIOMEDICAL IMPORN\NCE the pH of extracellular Muid bgyef€en 7.35 and 745. Suspected Water is the predominant chemical co organisms, Its unique physical properties, food pH <7.36) include diabetic ketosis ability to solvate a wide range of organic and ‘Alkalosis (pH >7.45) may follow vomiting ecules, derive from water's dipolar structure anc water interacts with a solvated biomolecule influences structure both of the biomolecule and of water itself, An excel~ Jent nucleophile, water isa reactant or product in many met- abolic reactions. Regulation of water balance depends upon, hypothalamic mechanisms that control thirst, on antidiury hormone (ADH), on retention or excretion of water ‘kidneys, and on evaporative loss. Nephrogenic diabet le isan irregular, slightly skewed tetrahedron with oxygen at We center (Figure 2-1). The two hydrogens and the unshared elekpns of the remaining two sp'-hybridized orbitals occupy the CMegers of the tetrahedron. The 105° angle ‘Water has a slight propensity to dissgfate into hydroxide between the two hydrogSggtoms differs slightly from the ideal ions and protons. The concentration ffrotons, or acidity, of tetrahedral angle, 109.5°. AMkggonia is also tetrahedral, with a aqueous solutions is generally repoyd using the logarithmic 107° angle between its three hy Magen. The strongly electro- pH scale. Bicarbonate and otherfuffers normally maintain negative oxygen atom in a water molecule attracts electronsWATER IS AN EXCELLENT NUCLEOPHILE ‘nucleophile. Other nucleophiles of biologici the oxygen atoms of phosphates, alcohols, an the sulfur of thiols; and the nitrogen atom of Amines and of the imidazole ring of histidine. Common electypphiles include the carbonyl carbons in amides, esters, aldehydes, and ketones and the phosphorus atoms of phosphoesters. Nucleophilic attack by water typicafy results in the cleav- age of the amide, glycoside, or ester byftds that hold biopoly- ‘mers together. This process is termedaydrolysis. Conversely, ‘when monomer units are joined tagether to form biopoty- ‘mers, such as proteins or glycogeh, water is a product, for example, during the formation of fpeptide bond between two amino acids While hydrolysis is a the tion, the amide and phosphoe; oligonucleotides are stable i dynamically favored reac: + bonds of polypeptides and the aqueous environment of the cell. This seemingly parafloxical behavior reflects the fact that the thermodynamics that govern the equilibrium point of a reaction do not deterfaine the rate at which it will pro- ceed toward its equilibriym point. In the cell, protein cata- lysts called enzymes accglerate the rate of hydrolytic reactions when needed. Proteasés catalyze the hydrolysis of proteins {nto their component/amino acids, while mucleases catalyze the hydrolysis of thefphosphoester bonds in DNA and RNA. Careful control of te activities ofthese enzymes is required to censure that they agf only at appropriate times. p Transfer wrymic reactions responsible for synthesis and biomolecules involve the transfer ofa chemical donor D to an acceptor A to form an acceptor ex, A Many of the breakdown covglent bonds strongly favors the formation of spit products. {versely, many group transfer reactions responsible for the bidsynthesis of macromolecules involve the thermodynami- coflly unfavored formation of covalent bonds, Enzyme catalysts Play a critical role in surmounting these barriers by virtue of, their capacity to directly link two normally separate reactions together. By linking an energetically unfavorable group transfer reaction with a thermodynamically favorable reaction, such as cHAPTER? voterap 9 the hydrolysis of ATP, a new coupled react ated whose net overall change in free ene synthesis. Given the nucleophilic charagi@ of water and its high concentration in cells, why are pfopolymers such as proteins and DNA relatively stable? Apéhow can synthesis of biopoly- mers occur in an aqueous esironment that favors hydrolysis? Central to both questionf are the properties of enzymes. In the absence of enzymigatalysis, even reactions that are highly favored thermodyngahically do not necessarily take place rap- idly. Precise and gifferential control of enzyme activity and the sequestration gfenzymes in specific organelles determine the physiologic Arcumstances under which a given biopolymer fhesized or degraded, The ability of enzyme active Aequester substrates in an environment from which ‘can be excluded facilitates biopolymer synthesis. Water Molecules Exhibit a Slight bt Important Tendency to Dissociate 1¢ ability of water to ionize, while slight, is of central impor- Tance for life. Since water can act both asan acid and as a base, its ionization may be represented as an intermolecular proton twansfer that forms a hydonium ion (H,O°) and a hydroxide ion (OH) ‘ean be gener- favors biopolymer H,0+H,02H,0+0H" ‘The transferred proton is actually associated with a cluster of ‘water molecules. Protons exist in solution not only as H,0°, but also as multimers such as H,O,” and HO,’ The proton is nevertheless routinely represented as H-, even though itis in fact highly hydrated. Since hydronium and hydroxide ions continuously recom- bine to form water molecules, an individual hydrogen or oxygen cannot be stated to be present as an ion or as part of a water molecule. At one instant itis an ion; an instant later it is part ofa water molecule. Individual ions or molecules are therefore not considered. We refer instead to the probability that at any instant in time a given hydrogen will be present as an ion owas part of water molecule. Since 1g of water contains 3.46 x 10 ‘molecules, the ionization of water can be described statisti= cally. To state that the probability that a hydrogen exists as an ion is 0.01 means that at any given moment in time, a hydro- {gen atom has 1 chance in 100 of being an ion and 99 chances ‘ut of 100 of being part of a water molecule. The actual prob- ability of a hydrogen atom in pure water existing as @ hydrogen ion is approximately 1.8 x 10°. The probability of its being part of a water molecule thus is almost unity. Stated another ‘way, for every hydrogen ion or hydroxide ion in pure water, there are 0.56 billion or 0.56 x 10° water molecules. Hydrogen ions and hydroxide ions nevertheless contribute significantly to the properties of water. For dissociation of water, [HOH] {H,0)10 SECTION! structs Function: often Enzymes where the brackets represent molar concentrations (strictly speaking, molar activities) and K is the dissoci Since 1 mole (mol) of water weighs 18°g, I liter (L) (1000 g) of water contains 1000 + 18 = 55.56 mol. Pure water thus is 55.56 molar. Since the probability that a hydrogen in pure ‘water will exist as a hydrogen ion is 1.8 10%, the molar con- centration of H" ions (or of OH" ions) in pure water is the product of the probability, .8 x 10%, times the molar concen- {ration of water, $5.56 moV/L. The result is 1.0.x 10° mol/L. ‘We can now calculate the dissociation constant K for pure water: tur (Orr) _ (107107) THO] 15556] =0.018x10- =1.8x10 mol/L. The molar concentration of water, 55,56 mol/L is too great to be significantly affected by dissociation. Itis therefore consid- ered to be essentially constant. This constant may therefore be incorporated into the dissociation constant K to provide a useful new constant K, termed the ion product for wate. The felationshp between K and Ks skoaR BSR {HOH 10° mol. THO] K)[H,0]=(H° 017} (18x10 mol/L)(55:56molL) .00%10-**(molLY Note that the dimensions of K are moles per liter and those of K,, are moles? per liter®, As its name suggests the ion product Kis numerically equal to the product of the molar concentra- tions of H* and OH =[H" OH] At 25°C, K = (10°), or 10" (mol/L). At temperatures below 25°C, K, is somewhat less than 10", and at tempera- tures above 25°C itis somewhat greater than 10°. Within the stated limitations of temperature, K, equals 10-™ (mol/L)? for all aqueous solutions, even solutions of acids or bases. We use K ,to calculate the pH of acidic and basic solutions. PH IS THE NEGATIVE LOG OF THE HYDROGEN ION CONCENTRATION ‘The term pH was introduced in 1909 by Sorensen, who defined itas the negative log of the hydrogen ion concentration: pH=loglH"] This definition, while not rigorous, suffices for many bio- chemical purposes. To calculate the pH of a solution: 1, Calculate the hydrogen ion concentration [H*] 2, Caleulate the base 10 logarithm of (1°). 3. pHTis the negative of the value found in step 2. For example, for pure water at 25°C, pH=-logltt ogo” (-7) ‘This value is also known as the power (English), puissant (French), or potenmz (German) of the exponent, hence the use ofthe term" Low pH values correspond to high concentrations of H* and high pH values correspond to low concentrations of 1 ‘Acids are proton donors and bases are proton acceptors. Strong acids (eg, HCL H,S0,) completly dissociate into anions earner crn in strongly acidic sobtions (ow pH). Weak dissociate only partially in acidic solutions, SimTarly. ‘st bases (eg, KOH, NaOH), but not weak bases like CHORT Ta conpletlydissoisted even at HE PFT-Many biochemicals are weak acids. Exceptions include phosphory- lated intermediites, whose phosphoryl group contains two dissociable protons, the fst of which i strongly acidic. “The fllowing examples illustrate how to calculate the pH of acidic and basic solutions. ‘Example I: What isthe pH ofa solution whose hydrogen ion concentration 32% 10" mol/L? 1og(3.2)-logt0™) 05440 . Example 2: What is the pH of solution whose hydroxide ion concentration is 4.0x 10" mol/L? We first definea quantity pOH that is equal to —loglOH"| and that may be derived from the definition of K: K,=[H' Jor ]=10"" Therefore, log(H1" + loglOH pH+pOH=14 “To solve the problem by this approach: (OH ]=40x10" pOH =-logiOH"] Jog(40x10") og(40)—log(t0~*) 0.50440 4Now ‘The examples above illustrate how the logarithmic pH scale facilitates recording and comparing hydrogen ion concentra- tions that differ by orders of magnitude from one another, 0.00032 M (pH 3.5) and 0.000000000025 M (pH 10.6) Example 3: What are the pH values of (a) 2.0 10 mol/L KOH and of (b) 2.0 x 10 mol/L KOH? The OH arises from two sources, KOH and water. Since pH is determined by the total [H"] (and pOH by the total [OH')), both sources must be considered. In the first case (a), the contribution of water to the total [OH] is negligible, The same cannot be said for the second case (b) ‘Molantyof KOH 20105 20x10 [0H from KOH 20107 20104 [081 fom water 10x10 10x10? Total [OH] 2.00001 10 2ax10+ Oncea decision has been reached about the significance of the contribution by water, pH may be calculated as above. ‘The above examples assume that the strong base KOH is completely dissociated in solution and that the concentra- tion of OH" ions was thus equal to that due to the KOH plus that present initially in the water. This assumption is valid for dilute solutions of strong bases or acids, but not for weak bases of acids. Since weak electrolytes dissociate only slightly in solution, we must use the dissociation constant to caleu- late the concentration of [11] (or [OH produced by a given ‘molarity of a weak acid (or base) before calculating total [H"] (or total [OF] and subsequently pH. \ctional Groups That Are Weak Acids lave Great Physiologic Significance Many biochemicals possess functional groups that are weak acids or bases. Carboxyl groups, amino groups, and phosphate esters, whose second dissociation falls within the physiologic range, are present in proteins and nucleic acids, most coen- zymes, and most intermediary metabolites. Knowledge of the dissociation of weak acids and bases thus is basic to under- standing the influence of intracellular pH on structure and bio- logic activity. Charge-based separations such as electrophoresis and ion exchange chromatography are also best understood in terms of the dissociation behavior of functional groups. ‘We term the protonated species (HA or R—NH,’) the acid and the unprotonated species (A~ or R—NH)) its conjugate base. Similarly, we may refer to a base (A" or R—NH,) and its conjugate acid (HA or R-NH,’). COPTER? Water u ‘We express the relative strengths of weak acids and bases, in terms of their dissociation constants, Shown below are the ‘expressions for the dissociation constant (K) for two repre- sentative weak acids, R-COOH and R—NH” R—COOH =2R—COO-+H* [R=COO"}[HH") *" [R—COOH] RONH,' @2R—NH, +H" [R=NH, JH] [R—NE,'] Since the numeric values of K, for weak acids are negative ‘exponential numbers, we express K, as pK, where PK, =-logk, ‘Note that pK, is related to K, as pH isto [H']. The stronger the acid, the lower is its pK, value. Representative weak acids (left), their conjugate bases (center), and pK, values (right) include the following: R—CH,—COOH —- R—CH,COO™ ROCH,—NH = RCH,—NH, pk, H,C0, HCO, Pk, HPO” PK, DK, is used to express the relative strengths of both acids and bases. For any weak acid, is conjugate isa strong base. Similarly, the conjugate of a strong base is a weak acid. The relative strengths of bases are expressed in terms of the pK, of their ‘conjugate acids. For polyprotic compounds containing more than one dissociable proton, a numerical subscript is assigned to each dissociation, numbered starting from unity in decreas- ing order of relative acidity. Fora dissociation of the type RONH} 9R—NH, +H" the pK, is the pH at which the concentration of the acid R— NH," equals that of the base R-NH,, From the above equations that relate K, to [H'] and to the concentrations of undissociated acid and its conjugate base, when {R—COO }=[R—COOH]) or when (RN, ]=[R—NH] then K-18] ‘Thus, when the associated (protonated) and dissociated (conjugate base) species are present at equal concentrations,12 SECTION! structures Function: often Enzymes the prevailing hydrogen ion concentration [H*| is numerically equal to the dissociation constant, K, Ifthe logarithms of both sides ofthe above equation are taken! and both sides are multi- plied by -1, the expressions would be as follows: K=[8"] log k,=loglH"] Since “log K, is defined as pK, and log {H"| defines pH, the equation may be rewritten as, pK,=pH that is, the pK, of an acid group is the pH at which the pro- tonated and unprotonated species are present at equal con- centrations, The pK, for an acid may be determined by adding 05 equivalent of alkali per equivalent of acid. The resulting 1H will eqyal the pK, of the acid uation Describes the Behavior of Weak Acids & Buffers ‘The Henderson-Hasselbalch equation is derived below. ‘Aweak acid, HA, ionizes as follows: Hace EA" ‘The equilibrium constant for this dissociation is (AT Tay Cross-multiplication gives (1A ]=K,(HA) Divide both sides by [A“} (HA] I }=K, ‘Take the log of both sides: ltt) ia (Hal =logk, Hog 4! BK PBT] Multiply through by Z (al ~loglit"|=ogk, -Iog!24) lH] oe, ig Substitute pH and pK, for —log [}1"] and “log K, respectively; then soglttAl = pK, -1 PH=PK, “log jenderson-Hasselbalch Denivolic 0K Inversion of the last term removes the minus sign and gives the Henderson-Hasselbalch equation [A] i= pK, tog AL Pare 8 THA ‘The Henderson-Hasselbalch equation has great predictive value in protonic equilibria. For example, 1, When an acid is exactly half-neutralized, [47] = [HA] Under these conditions, = pK, +log | - px, slog} J=pK, +4 PH=pK, tog A =, +g 2} PK, +0 Therefore, at half-neutralization, pH = pK, io [A-Y/(H1A] = 100:1, 1x1 THA] pH=pK, +1og(100/1)= 2. When th pH=pK, +log. 3. When the ratio [A"/[HA]= 1:10, pH=pK, + log(1/10)= px, +(-1) If the equation is evaluated at ratios of [A-V/[HA] ranging from 10° to 10-" and the calculated pH values are plotted, the resulting graph describes the titration curve for a weak acid (Figure 2-5). Solutions of Weak Acids & Their Salts Buffer Changes in pH Solutions of weak acids or bases and their conjugates exhibit bufiering, the ability to resist a change in pH following addi tion of strong acid or base. Many metabolic reactions are accompanied by the release or uptake of protons. Oxidative metabolism produces CO,, the anhydride of earbonic acid, which if not buffered would produce severe acidosis. Biologic ‘maintenance of a constant pH involves buffering by phosphate, bicarbonate, and proteins, which accept or release protons to Bon os g boo eg Boe og fos ~02 Fo ° IE 2-5 Titration curve for an acid of the type HA. heavy dot inthe center of the curve indicates the pk, 5.0.resist a change in pH. For laboratory experiments using tissue extracts or enzymes, constant pH is maintained by the addi- tion of buffers such as MES ([2-N-morpholino|-ethanesulfonic acid, pK, 6.1), inorganic orthophosphate (pK,, 7.2), HEPES (-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, pK, 68), or Tris (trishydroxymethyljaminomethane, pK, 8.3). The value of pK, relative to the desired pH is the major determinant of, which butfer is selected. Buflering can be observed by using a pH meter while titrating a weak acid or base (Figure 2=5). We can also cal- culate the pH shift that accompanies addition of acid or base to a buffered solution. In the example below, the buffered solution (a weak acid, pK, = 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: initia pH 500537560586 Ung on ooo ‘, os oe WOM, 02334807 EE ee a a ae ti ce as LASTS Fae a regs sa Tas Ta oon oo ‘a Notice that ApH, the change in pH per milliequivalent of OH" added, depends on the initial pH. The sofution resists changes in pH most effectively at pH values close to the px, A solution of a weak acid and its conjugate base buffers ‘most effectively in the pH range pK, * 1.0 pH unit. Figure 2-S also illustrates how the net charge on one mole- cule ofthe acid varies with pH. A fractional charge of -0.5 does ‘not mean that an individual molecule bears a fractional charge but that the probability is 0.5 that @ given molecule has a unit negative charge at any given moment in time, Consideration of the net charge on macromolecules asa function of pH pro- vides the basis for separatory techniques such as ion exchange chromatography and electrophoresis (see Chaptgr 4). Acid Strength Depends on Molecular Structure ‘Many acids of biologic interest possess more than one dissoci- ating group. The presence of local negative charge hinders pro- ton release from nearby acidic groups, raising their pK, This is illustrated by the pK, values of the three dissociating groups of phosphoric acid and citric acid (Table 2-2). The effect of, adjacent charge decreases with distance. The second pK, for CuNPTER? Water 13 ‘TABLE 2-2 Relative Strengths of Selected Acids of Biologic Significance Formic ek 375 Lacie pr 386 eatic pk 476, ‘Ammonium fon PK 928 Carbon -% “a7 2 1028 soci ™* ai - sea arc -% a3 0 sai iret Phosphoc ro 25 oa tase cme 6 200 - a sao Not: Tabulates values ae the pK, values (og ofthe dssocatonconstand ot ‘Miecied monopratic dpretc and Wipe as succinic acid, which has two methylene groups between its carboxyl groups, is 5.6, whereas the second pK, for glutaric acid, which has one additional methylene group, is 54 pK, Values Depend on the Properties of the Medium ‘The pK, of a functional group is also profoundly influenced by the surrounding medium. The medium may either raise ‘or lower the pK, relative to its value im water, depending on Whether the undissociated acid or its conjugate base is the charged species. The effect of dielectric constant on pK, may bbe observed by adding ethanol to water. The pK, of a catbox- yilic acid increases, whereas that of an amine decreases because ‘ethanol decreases the ability of water to solvate a charged spe cies. The pK, values of dissociating groups in the interiors proteins thus are profoundly affected by their local environ. ‘ment, including the presence or absence of water. SUMMARY Water forms hydrogen-bonded clusters other proton donors or acceptors. Hydrogen bonds account for the surface tension, viscosity liquid state at room temperature, and solvent power of water6 1. Amino Acids lll. ACIDIC AND BASIC PROPERTIES OF AMINO ACIDS Amino acids in aqueous solution contain weakly acidic a-carboxy! ‘groups and weakly basic a-amino groups. In addition, each of the acidic, and basic amino acids contains an ionizable group in its side chain Thus, both free amino acids and some amino acids combined in peptide linkages can act as buffers. Recall that acids may be defined as proton donors and bases as proton acceptors. Acids (or bases) described as ‘weak’ ionize to only a limited extent. The concentration of protons in aqueous solution is expressed as pH, where pH = log 1/{H"] or —log [H*]. The quantitative relationship between the pH of the solu- tion and concentration of a weak acid (HA) and its conjugate base (A") is described by the Henderson-Hasselbalch equation. A. Derivation of the equation Consider the release of a proton by a weak acid represented by HA: HA 2 H+ weak proton salt form acid or conjugate base 3. The “salt” or conjugate base, A-, is the ionized form of a weak acid, By definition, the dissociation constant of the acid, Ka, is k, = HAT = THA [Note: The larger the Ka, the stronger the acid, because most of the HA has dissociated into H* and A’. Conversely, the smaller the Ka, the less acid has dissociated and, therefore, the weaker the acid.) By solving for the [H'] in the above equation, taking the logarithm of both sides of the equation, multiplying both sides of the equation by =1, and substituting pH = —log [H* ] and pKa = —log Ks, we obtain the Henderson-Hasselbalch equation: Titration curve of deetic acid. PH = pK + 109 [Fal B. Buffer A buffer is a solution that resists change in pH following the addition of an acid or base. A butter can be created by mixing a weak acid (HA) with its conjugate base (A°). If an acid such as HCI is then added to such a solution, A can neutralize it, in the process being converted to HA. If a base is added, HA can neutralize it, in the process being converted to ‘AC. Maximum buffering capacity occurs at a pH equal to the pKa, but a conjugate acid/base pair can stil serve as an effective buffer when the pH of a solution is within approximately =1 pH unit of the pK, If theIll. Acidic and Basic Properties of Amino Acids Figure 1.10 lonic forms of alanine in acidic, neutral, and basic solutions, amounts of HA and A~ are equal, the pH is equal to the pK. As shown in Figure 1.9, a solution containing acetic acid (HA = CH,—-COOH) and acetate (A” = CHs~COO>) with a pK. of 4.8 resists a change in pH trom pH 3.8 to 5.8, with maximum buffering at pH 4.8. At pH values less than the pKa, the protonated acid form (CHs~COOH) is the predominant species. At pH values greater than the pK, the deprotonated base form (CHs-COO>) is the predominant species in solution. C. Titration of an amino acid 1, Dissociation of the carboxyl group: The titration curve of an ‘amino acid can be analyzed in the same way as described for acetic acid. Consider alanine, for example, which contains both an a-carboxyl and an camino group. At a low (acidic) pH, both of these groups are protonated (shown in Figure 1.10). As the pH of the solution is raised, the -COOH group of Form | can dissociate by donating a proton to the medium. The release of a proton results in the formation of the carboxylate group, COO . This structure is shown as Form Il, which is the dipolar form of the ‘molecule (see Figure 1.10). This form, also called a zwitterion, is the isoelectric form of alanine, that is, it has an overall (net) charge of zero. 2. Application of the Henderson-Hasselbaich equation: The dissoci- ation constant of the carboxyl group of an amino acid is called Ki, rather than Ka, because the molecule contains a second titratable group. The Henderson-Hasselbaich equation can be used to analyze the dissociation of the carboxyl group of alanine in the same way as described for acetic acid: _ wa "1 where | is the fully protonated form of alanine, and II is the iso- electric form of alanine (see Figure 1.10). This equation can be rearranged and converted to its logarithmic form to yield:8 1. Amino Acids 3, Dissociation of the amino group: The second titratable group of alanine is the amino (~NHs") group shown in Figure 1.10. This is a much weaker acid than the -COOH group and, therefore, has a ‘much smaller dissociation constant, Ko. [Note: Its pK is therefore larger Release of a proton from the protonated amino group of Form Il resuits in the fully deprotonated form of alanine, Form Ii (see Figure 1.10). 4. pKs of alanine: The sequential dissociation of protons from the carboxyl and amino groups of alanine is summarized in Figure 1.10. Each titratable group has a pK that is numerically equal to ame a the pH at which exactly one half of the protons have been removed from that group. The pK for the most acidic group (-COOH) is pK;, whereas the pK, for the next most acidic group (ENHg') is pK, 5. Titration curve of alanine: By applying the Henderson- Hasselbalch equation to each dissociable acidic group, itis possi ble to calculate the complete titration curve of a weak acid. Figure 1.11 shows the change in pH that occurs during the addition of base to the fully protonated form of alanine (|) to produce the completely deprotonated form (Il). Note the following: . Buffer pairs: The -COOH/-COO™ pair can serve as a butter in the pH region around pK;, and the -NHs"/-NHp pair can butfer in the region around pke. b. When pH = pK: When the pH is equal to pk; (2.3), equal amounts of Forms | and I of alanine exist in solution, When the pH is equal to pk (9.1), equal amounts of Forms Il and Ill are present in solution Figure 1.11 neutral pH, alanine exists predominantly The titration curve of alanine. rm II in which the amino and carboxyl groups are ionized, but the net charge is zero. The isoelectric point (p!)is the pH at which an amino acid is electrically neutral, that is, in which the sum of the positive charges equals the sum of the negative charges. For an amino acid, such as alanine, that has only two dissociable hydrogens (one from the c-carboxyl and one from the a-amino group), the pl is the average of pK: and pk (pl = [2.3 + 9.1)/2 = 5.7, see Figure 1.11). The pl is thus midway between pK; (2.3) and pK; (9.1). pl corresponds to the pH at which the Form Il (with a net charge of zero) pre- dominates, and at which there are also equal amounts of Forms | (net charge of +1) and lll (net charge of 1). seen ontone4 Ill, Acidic and Basic Properties of Amino Acids Separation of plasma proteins by charge typically is done at a pH above the pl of the major pro- ‘eins, thus, the charge.on the proteins is negative. In an electric field, the proteins will move toward the positive electrode at a rate determined by their net negative charge. Variations in the mobil- ity pattern are suggestive of certain diseases. 6. Net charge of amino acids at neutral pH: At physiologic pH, amino acids have a negatively charged group (-COO") and a positively charged group (~NHs*), both attached to the a-carbon [Note: Glutamate, aspartate, histidine, arginine, and lysine have additional potentially charged groups in their side chains.] ‘Substances, such as amino acids, that can act either as an acid or a base are defined as amphoteric, and are referred to as ampholytes (amphoteric electrolytes). D. Other applications of the Henderson-Hasselbaich equation ‘The Henderson-Hasselbalch equation can be used to calculate how the pH of a physiologic solution responds to changes in the concen- tration of a weak acid and/or its corresponding “salt” form. For exam- ple, in the bicarbonate buffer system, the Henderson-Hasselbalch equation predicts how shifts in the bicarbonate ion concentration, [HCOs"], and COz influence pH (Figure 1.12A). The equation is also useful for calculating the abundance of ionic forms of acidic and basic drugs. For example, most drugs are either weak acids or weak bases (Figure 1.12B). Acidic drugs (HA) release a proton (H*), caus- ing a charged anion (A°) to form HA OHTHA Weak bases (BH") can also release a H*. However, the protonated form of basic drugs is usually charged, and the loss of a proton pro- duces the uncharged base (B). BH = B+ A drug passes through membranes more readily if it is uncharged. Thus, for a weak acid such as aspirin, the uncharged HA can per- meate through membranes and A” cannot. For a weak base, such as morphine, the uncharged form, B, penetrates through the cell membrane and BH* does not. Therefore, the effective concentration of the permeable form of each drug at its absorption site is deter- mined by the relative concentrations of the charged and uncharged forms. The ratio between the two forms is determined by the pH at the site of absorption, and by the strength of the weak acid or base, which is represented by the pK, of the ionizable group. The Henderson-Hasselbalch equation is useful in determining how much drug is found on either side of a membrane that separates two com- partments that differ in pH, for example, the stomach (pH 1.0-1.5) and blood plasma (pH 7.4). ‘causes the pH to rise. Pulmonary obstruction causes an| increase in carbon dioxide and ‘causes the pH to fall, resulting In respiratory acidosis. (97 Bithe pH ofthe stomach (1.5),a ‘rug ke aspirin (weak acio, pk = 35) wil be largely protonated eee Tncharged arugs: snes more rapidly than HA SscS = § =< LuMEN OF = STOMACH Figure 1.12 ‘The Henderson-Hasselbalch equation is used to predict: A, changes in pH as the concentrations ‘of HCO5" or COz are altered; or B, the ionic forms of drugs.Kelevance > Marks \ Biiffers. A buffer is a mixture of an undissociated acid and its conjugate base (the form of the acid having lost its proton). It causes a solution to resist changes in pH when either H* or OH” is added. A buffer has its greatest buffering capacity in the pH range near its pK, (the negative log of its K,). Two factors determine the effectiveness of a buffer, its pK, relative to the pH of the solution and its concen- tratior — Metabolic Acids and Bases. Normal metabolism generates CO, metabolic acids (e.g., lactic acid and ketone bodies) and inorganic acids (e.g., sulfuric acid). The major source of acid is CO,, which reacts with water to produce carbonic acid. To maintain the pH of body fluids in a range compatible with life, the body has buffers such as bicarbonate, phosphate, and hemoglobin (see Fig 4.1). Ulti- mately, respiratory mechanisms remove carbonic acid through the expiration of CO,, and the kidneys excrete acid as ammonium ion (NH,") and other ions.