BIOMOLECULES (INTRODUCTION, STRUCTURE & FUNCTIONS)

Amino Acids

Abbas Ali Mahdi

Professor Department of Biochemistry

King George’s Medical University Lucknow - 226 003, India.

22-May-2007 (Revised 11-Sep-2007)

CONTENTS

IntroductionCommon structural features of amino acidsStereoisomerism of amino acidsRS system of designating optical isomers Compounds having two or more stereogenic centersClassification and structure of standard amino acidsZwitterion in aqueous solutionPhysical and chemical properties of amino acidsTitration of amino acidsSeparation of amino acidsEssential amino acidsWater as biological solventWeak acids and basespHBuffersHenderson Hasselbalch equationPhysiological buffersFitness of the aqueous environment for living organisms Keywords Amino acid; Isomerism; Zwitterion; Water; pH; Buffer

Introduction

Protein, the term is derived from Greek word Proteios means “ primary” or “holding first place” or “pre eminent” because Jons Jacob Berzelius, a Swedish chemist, thought them to be most important of biological substances. And now we know that proteins are fundamental structural and functional components of the body. They are nitrogenous “macromolecules” composed of many amino acids. Most proteins contain, in varying proportions, the same 20 L-α-amino acids. Many specific proteins contain, in addition, L-α-amino acids derived from some of the basic 20 amino acids by processes that occur after formation of the polypeptide backbone. These unusual amino acids fulfill highly specific functions for the protein and increase biologic diversity. This chapter considers the structures, physical properties, stereochemistry, chemical properties and ionic equilibrium of the amino acids. Also considered are methods for separation and the importance of water in our life. Common structural features of amino acids

Basic structure of an amino acid is depicted in Fig 1. All the 20 amino acids have a carboxyl group and an amino group attached to the α-carbon.

R C COOH

H

NH2

Fig. 1.

In case of glycine, R is H, while in rest of the amino acids it varies, e.g. alanine has a methyl (CH3) side chain group. Amino acids are ordinarily designated by three letter abbreviations but a set of single letter symbols is also adopted, as shown in the Table 1.

Stereoisomerism of amino acids

All amino acids, except glycine, show optical activity i.e., they can rotate the plane polarized light. Optical activity is exhibited by all compounds that are capable of existing in the forms that are non-superimposable mirror images of each other; such compounds are called chiral compounds. This phenomenon of stereoisomerism is called chirality. This occurs in all compounds having an asymmetric carbon atom i.e., carbon atom having four different substituents, optical activity is expressed as the specific rotation [ ] : 25

Dα

[ ])/.()(

100)(25

dLgconcdmlengthpathopticalrotationobserved

D ××

=οα

2

Table 1: Amino acid symbols

Amino acid Three-letter abbreviation One letter symbol Alanine Ala A Arginine Arg R Asparagine Asn N Aspartic acid Asp D Cysteine Cys C Glutamine Gln Q Glutamic acid Glu E Glycine Gly G Histidine His H Isoleucine Ile I Leucine Leu L Lysine Lys K Methionine Met M Phenylalanine Phe F Proline Pro P Serine Ser S Threonine Thr T Tryptophan Trp W Tyrosine Tyr Y Valine Val V

Some α-amino acids isolated from proteins are dextrorotatory (Ala, Ila, Glu etc.) i.e., right handed, while others are levorotatory (Trp, Leu, Phe) i.e., left handed. Dextrorotatory compounds are designated with [+] and levorotatory compounds with [–]. The specific rotation of an amino acid varies with the pH, and also depends on the nature of its R group. The stereochemistry of amino acids is discussed in terms of the absolute configuration of the four different substituents in the tetrahedron around the asymmetric carbon atom. The compound best selected to serve as a standard to explain stereoisomer is three C sugar glyceraldehydes, which has an asymmetric carbon atom. Two possible stereoisomers of glyceraldehyde are designated as L and D. The relationship of the amino acid alanine with glyceraldehyde is discussed below (Fig. 2). CHO CHO HO C H H C OH CH2OH CH2OH L-Glyceraldehyde D-Glyceraldehyde

3

COO- COO-

H3N+ C H H C +NH3

CH3 CH3 L-Alanine D-Alanine

Fig. 2 Here, we observe that the amino group on the asymmetric carbon atom of alanine can be stereochemically related to the substituent hydroxyl group on the asymmetric carbon atom of glyceraldehyde, the carboxyl group of alanine can be related to the aldehyde group of glyceraldehyde, and the R group of alanine can be related to the –CH2OH group of glyceraldehyde. Thus, isomers stereochemically related to L-glyceraldehyde are designated L, and those related to D-glyceraldehyde are designated D, regardless of the direction of rotation of plane polarized light. The symbols D and L thus refer to absolute configuration, not direction of rotation. The D and L stereoisomers of any compound have identical physical properties and identical chemical reactivity, with two exceptions:

i. They rotate the plane of polarized light equally but in opposite directions. ii. They react at different rates with symmetric and asymmetric reagents.

The amino acids, which occur naturally belong to L stereochemical series. The amino acids with two asymmetric carbon atoms, threonine and isoleucine, have four stereoisomers, here the other two stereoisomers are called diastereoisomers or alloisomers. RS system of designating optical isomers

RS system of designating optical isomers was proposed in 1956, by three European chemists; Robert D Cahn, Christopher K Ingold and Valadimir Prelog. This system of nomenclature is also known as CIP system or the R-S system. In this system, each stereogenic center in a moleculer is assigned a prefix (R or S). The symbol R is derived from latin word ‘rectus’ for right, and S from ‘sinister’ for left. The assignment of these prefixes depends on the application of two rules: The sequence rule and the viewing rule. The Sequence Rule

The rule has following assumptions; (1) Assign sequence priorities to the four substituents by looking at the atoms

attached directly to the chiral carbon atom. (2) The higher the atomic number of the immediate substituent atom, the higher the

priority. eg. H<C<N<O<Cl.

(3) If two substituent’s have the same immediate substituent atom, evaluate the atoms progressively further away from the chiral center until a difference is found.

4

eg. CH3< C2H5< ClCH2 <BrCH2< CH3O. (4) If double or triple bonded groups are encountered as substituents, they are

treated as an equivalent atom. eg. C2H5- < CH2 =CH-

The Viewing Rule

Once the priorities of all the four substituents have been determined, the chiral center must be viewed from the side opposite the lowest priority group. If we number the substituent groups from 1 to 4, with, 1 being the highest and 4 the lowest in priority sequence, the two enantiomeric configurations are shown in the following figure (3) along with viewer eye on the side opposite substituent 4.

Fig. 3

Here an observer notes whether a curved arrow drawn from the # 1 position to the # 2 location and then to the # 3 position turns in a clockwise or counter-clockwise manner. If the turn is clockwise, the configuration is classified R. If it is counter-clockwise, the configuration is S. Here, it is important to remember that there is no simple or obvious relationship between the R or S designation of a molecular configuration and the experimentally measured specific rotation of the compound it represents. Compounds having two or more stereogenic centers

The shrub Ephedra vulgaris contains two physiologically active compounds ephedrine and pseudoephedrine. Both compounds are stereoisomers of 2-methylamino-1-phenyl-1-propanol, and both are optically active, one being levorotatory and the other dextrorotatory (Fig. 4). Since these two compounds are optically active, each must have an enantiomer. Although these missing stereoisomers were not present in the natural source, they have been prepared synthetically and have the expected identical physical properties. The structural formula of 2-methylamino-1-phenylpropanol has two stereogenic carbons (1 & 2). Each may assume an R or S configuration, so there are four stereoisomeric combinations.

Fig. 4

5

Stereogenic nitrogen atom

The ephedrine and pseudoephedrine isomers suggest that another stereogenic center, the nitrogen, is present. Here single-bonded nitrogen is pyramidal in shape, with the non-bonding electron pair pointing to the unoccupied corner of a tetrahedral region. Since the nitrogen in these compounds is bonded to three different groups, its configuration is chiral. Fig. 5 illustrates the mirror image configurations.

Fig. 5 If these configurations were stable, there would be four additional stereoisomers of ephedrine and pseudoephedrine. The pyramidal nitrogen is normally not configurationally stable. It rapidly inverts its configuration by passing through a planar, sp2-hybridized transition state, leading to a mixture of interconverting R and S configurations. Classification and structure of standard amino acids

Amino acids are classified on the basis of their R groups i.e., based on their polarity. The four main classes are:

1. Non-polar R groups. 2. Polar R groups. 3. Positively charged R groups. 4. Negatively charged R groups.

1. Amino Acid with Non-polar R Groups

This family includes eight amino acids, including five with aliphatic R groups (Alanine, leucine, isoleucine, valine and proline), two with aromatic rings (phenylalanine and tryptophan), and one containing sulfur (methionine) as shown in Fig. 6. These are less soluble in water as compared to polar R group containing amino acids. Moreover, glycine is also a non-polar amino acid, which has a hydrogen, instead of any functional group. 2. Amino Acids with Uncharged Polar R Groups

The amino acids of this family contain neutral polar functional groups in their R groups. This family includes six amino acids. Three amino acids contain hydroxyl group (serine, threonine and tyrosine), two contain amide group (asparagine and glutamine) while one contain sulfhydryl group (cysteine), responsible for polarity (Fig. 7).

6

Fig. 6: Amino acids with non-polar R groups.

Fig. 7: Amino acids with polar R groups.

3. Amino Acids with Positively Charged R Groups

The amino acids of this family are basic in nature. The R group of these has a net positive charge at pH 7.0, all have six carbon atoms. This family of amino acids consist of lysine, which possess a positively charged amino group at ε position on aliphatic chain; arginine

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possess the positively charged guanidinium group; and histidine which contains weakly basic imidazolium functional group (Fig. 8).

Fig. 8: Amino Acids with Positively Charged R Group 4. Amino Acids with Negatively Charged R Groups

This family contains two members i.e., aspartic acid and glutamic acid,carboxylic group, which is fully ionized and thus negatively charged at 9).

Fig. 9: Amino Acids with Negatively Charged R Grou

In addition to these 20 standard amino acids, several other have rare ospecialized types of protein. These are derivatives of standard amino acid Example: 4-hydroxyproline, hydroxylysine, ε-N-methyllysine, methyl hisodesmosine, etc as shown in Box 1 and 2. Apart from these, over 300 amino acids are known to occur in free or never in proteins. Most of them are derivatives of L-α-amino acids buacids are also known (Box 3). These non-protein amino acids are imporintermediates during metabolic processes. For example, homocysteine aintermediates in amino acid metabolism, while citrulline and ornithine a

H +

s

each has a second pH 6.0 to 7.0 (Fig.

ps

ccurrence in some s.

istidine, desmosine,

combined form but t, β, γ and δ amino tant precursors and nd homoserine are re intermediates in

8

the synthesis of arginine and urea. γ-amino butyric acid (GABA) acts as chemical agent during transmission of nerve impulse.

H

OH

H

COOH

NH2CH2CHCH2CH2CHCOOH

OH NH2

CH3NHCH2CH2CH2CHCOOH

NH2

4-hydroxyproline 5-hydroxylysine ε-N-methyllysine

N NH3C

CH2CHCOOH

NH2

H

H

3

CH

COOHH2N

CH23

CH

NH2

COOH

CH2CH

H2N

HOOC

CH2 4

CH

H2N COOH

*

N+

CH2

2

3-methylhistidine Desmosine

Box 1

CH2 2CH

NH2

COOHCH2CH

H2N

HOOC

CH2 4

CH

H2N COOH

* *

N+ CH2 CH

NH2

COOH2

2

Isodesmosine

2

N H H

Box 2

9

CH2CH2CH2COOH

NH2

CH2CH2CHCOOH

SH NH2

CH2CH2CHCOOH

OH NH2

γ- aminobutyric acid Homocysteine Homoserine

CH2CH2CH2CHCOOH

NH2NH2

NHCH2CH2CH2CHCOOH

O NH2

H2N C

Ornithine Citrulline

Box 3 Zwitterion in aqueous solution

Many substances contain both acidic and basic groups, thus have capability to react with alkalies as well as acids to form salts. These type of substances are called ampholytes. Amino acids also possess this property. The property of amino acids as ampholytes can be understood with the example of simplest amino acid, glycine.

NH2

H C COOH

H

Here, -COOH is acidic group (proton donor), and the –NH2 is basic group (proton acceptor). In crystalline state, amino acid exists as zwitterion as a result of H+ ions passing from –COOH to –NH2 group.

NH3+

H C COO-

H

In acidic condition, zwitterions combines with H+ ions to form a cation, NH3

+

H C COOH

H

Which dissociates H+ ions in two stages;

10

+H3N-CH2COOH +H + +H3N-CH2-COO- +H +H2N-CH2-COO-

Cation Zwitterion Anion Thus, when NaOH is added to cationic solution of glycine, OH- ions first combine with H+ from the ionization of –COOH group and subsequently with H+ from –NH3

+ group to form H2O and glycine anion, while when HCl is added to glycine anion solution, H+ ions causes the formation of glycine cation. In conclusion, we can say, with NaOH the amino acid forms the sodium salt and with HCl it forms amino acid chloride. In the end, zwitterions can be defined as molecules that contain charged groups of opposite polarity (this exists only at pI). Physical and chemical properties of amino acids

Physical Properties: They are colorless, crystalline substances more soluble in water than in polar solvents. They have high melting point, usually more than 2000C. They have a high dielectric constant. They possess a large dipole moment. Chemical Properties: Reactions of the Carboxyl Group The carboxyl groups of amino acids may be esterified with alcohols

Or converted into the corresponding acyl chloride O

H+

R—CH—COOH + C2H5OH R—CH—C—OC2H5 + H2O

+NH3 +NH3

PCl5

R—CH—COO- R—CH—COClPOCl3

+NH3 +NH3

In the latter case the +NH3-group would first have to be protected to prevent its reacting (violently) with the PCl5. Such acyl chlorides represent activated forms of the amino acid, which in turn can be coupled with the amino group of a second amino acid to produce a dipeptide. The amide bond linking the two amino acids is known as the peptide bond:

H

—C—N—

O

Peptide bond

The properties of this substituted amide linkage play a unique role in determining the structure of proteins. A peptide containing two or more peptide bonds will react with Cu2+

11

in alkaline solution to form a violet-blue complex. This reaction, known as the “Biuret reaction”, is the basis for quantitative determination of proteins. The carboxyl group of amino acids may be decarboxylated chemically and biologically to yield the corresponding amine:

R—CH—COO- R—CH2 + CO2

NH3+ NH3

+

Amino acid amine

For example, the vasoconstrictor agent, histamine, is produced from histidine. Histamine stimulates the flow of gastric juice into the stomach and is also involved in allergic responses. Reactions of the amino group The amino group of an amino acid will react with the strong oxidizing agent nitrous acid (HNO2) to liberate N2. This reaction, which is stoichiometric, is important in the estimation of amino acids α-amino groups. The amino acids proline and hydroxyproline do not react, and the ε-amino group of lysine reacts, but at a slower rate. The products are the corresponding α-hydroxy acid and N2 gas, which can be measured manometrically. R—CH—COOH + HNO2 R—CH—COOH + N2 + H2O + H+

+NH3 OH

The amino group of amino acids will also undergo oxidation with the milder oxidizing agent ninhyrdin to form ammonia, CO2, and the aldehydes obtained by loss of one carbon from the original amino acid. In this reaction, one equivalent of ninhydrin serves as the oxidant for the amino acid to form the products just stated:

R—CH—COO- + Oxidized ninhydrin R—CH + NH3 + CO2 + Reduced ninhydrin

+NH3 O

A second equivalent of ninhydrin (oxidized) then reacts with the reduced ninhydrin and NH3 formed in the above equation to produce a highly colored product having the following structure:

C

C C C+ NH3 +

OH

O

O

HO C

O

Oxidized ninhy

OH H

drin Redu

C

O

ced ninhydrin

12

Blue product

The intense blue product is generally characteristic of amino acids having α-amino groups. However, proline and hydroxyproline, which are secondary amines, yield yellow products, and asparagines, which have a free amide group, reacts with ninhydrin to produce a characteristic brown product. The ninhydrin reaction is extensively employed in the quantitative determination of amino acids. Another reaction of the amino group which has found much recent use is the reaction with 1-fluoro-2,4-dinitrobenzene (FDNB):

In this reaction the intensely colored dinitrobenzene nucleus is attached to the nitrogen atom of the amino acid to yield a yellow derivative, the 2,4-dinitrophenyl derivative or DNP-amino acid. The compound FDNB will react with the free amino group on the NH2-terminal end of a polypeptide as well as the amino groups of free amino acids. Thus, by reacting a native protein or intact polypeptide with FDNB, hydrolyzing, and isolating the colored DNP-amino acid, one can identify the terminal amino acid in a polypeptide chain. The ε-amino group of lysine will also react with FDNB, but this derivative, ε-DNP-lysine, can readily be separated from the α-DNP-amino acids by an extraction procedure. The amino group of both free amino acids and peptide chains will react with dansyl chloride (5-dimethylamino-naphthalene-1-sulfonyl chloride) to produce a dansyl amino acid derivative. Because the dansyl group readily fluoresces, minute amounts of amino acids may be determined by this procedure.

Dansyl chlorid

O

C

CC + 3 H2O N=

H3N+—CH—COO- + NO2— —F NO2— —N—CH—CO2H + HF

R R H

NO2 NO2

N

H3N+—CH— COO- + R

SO2Cl

CH3 CH3

N

CH3 CH3

C

CC

OH

O

O

e Dansyl amino acid derivative

+ HCl

NH— CH—CO2H

SO2

R

13

The well-known reaction of isothiocyanates with amines has been ingeniously modified by Edman both to degrade a polypeptide chain and to identify the NH2-terminal amino acid in the peptide. Phenyl-isothiocyanate reacts with the α-amino group of an amino acid (or polypeptide) to form the corresponding phenylthiocarbamyl amino acid. In anhydrous acid, this compound cyclizes to form a phenylthiohydantoin, which is stable in acid.

Phenylthiohydantoin

If the NH2-terminal amino acid in a polypeptide is reacted with phenylisothio-cyanate and the derivative is subsequently treated with anhydrous acid, only the phenylthiohydantoin of the NH2-terminal amino acid is released. The remainder of the polypeptide chain remains intact, and this is usefulness of this method. The NH2-terminal group in the original peptide can of course be identified by determining the nature of the phenylthiohydantoin formed. Peptide synthesis The chemical synthesis of peptides can be carried out under controlled conditions that establish the order of the amino acids in the polypeptide. Such syntheses have been instrumental in establishing the structures of naturally occurring peptides and polypeptides. To synthesize a peptide, it is necessary to block the groups on the amino acid that can react during the synthesis of a peptide bond between two specific amino acids (or amino acid residue). Subsequently, the blocking group can be removed without destroying the newly synthesized peptide bond. If the synthesis is to join the carboxyl group of R1-CHNH2-COOH to the amino group of R2-CHNH2-COOH, the initial requirement is to block the amino group of the former. This can be done by reacting that amino acid with carbobenzoxy chloride.

Benzyl chlorocarbonate

—N=C=S + H3N+—CH— COO- —NH—C—NH—CH— COO-

R H+

R1—CHNH2—COOH + —CH2—O— C—Cl

O

CH2—O—C—NH—CH—CO2H + HCl

Benzyloxycarbonyl derivative

R1O

R S

S

N

NH

CH—R C

C

O

14

Next, the carboxyl group of that derivative must be reacted with the amino group of the second amino acid. However, in theory the carboxyl group of that amino acid should have first been blocked, perhaps as the benzyl ester.

O

R2—CHNH2—C—O—CH2— + H2O

Now, in order to couple the free carboxyl and amino group of the two derivatives, the carboxyl group (usually) must be activated. This could be by way of the acyl chloride. However, there are reagents such as dicyclohexylcarbodiimide (DCC) which can be used to activate and couple or condense in a single operation.

HN

Dip

Now, the protecting benzyl groups can be removed withoand the free dipeptide is formed.

O

O

R2—CHNH2—CO2H + —CH2OHH+

—CH2—O—C—NH—CH—CO2H + + R2—CHNH2—C—O—CH2—

R1

N

C

N

R2—C

OO R1

—CH2—O—C—N—CH—C

N

HH

NH C=O +

O=C

H

eptide Dicyclohexylurea

ut destruction of the peptide bond

NH CH2

15

Obviously, the blocked peptide could have been reacted with a third amino acid residue to form a tripeptide. This would usually require selective unblocking of either the amino or carboxyl group, depending on what synthesis was to be performed, followed by reaction of the unblocked functional group. The procedures just described have been used to synthesize countless peptides of known sequence, often by combining a series of small peptides to make a single large peptide. The procedure is extremely laborious and the yields of product are vanishingly small. In recent years, an imaginative synthesis of polypeptides on a solid phase as support has been devised. While the principles of blocking reactive groups, condensation, and unblocking are still employed, the novel use of an inert, insoluble polystyrene bead to bind the carboxyl group of the C-terminal amino acid permits filteration, washing and recovery of the product peptide on the bead. As before, reagents are used to unblock the N-terminal group, so that the next amino acid can be added, must be mild enough to avoid hydrolysis of peptide linkage, already formed as well as the bond to the resin. The solid phase procedure is rapid, the yields are high, and no racemization occurs. Simple oligopeptides and complete biologically active proteins have been synthesized. For example, bradykinin (9 residues) can be synthesized in less than a week. Insulin (51 residues), ferridoxin (55), acyl carrier protein (77), and even ribonuclease (129) have been effectively synthesized. Reactions of the R groups The ionization of the R groups of various amino acids have different physiological importance. R group of serine and cysteine have their biological importance in human body. The hydroxyl group of serine is present as phosphorylated form in a biologically active protein. For example, the glycolytic enzyme phosphoglucomutase contains a serine whose hydroxyl group becomes phosphorylated during the functioning of the enzyme. Also, the other example is milk protein casein, which contains a large number of phosphorylated serine residues.

. . . —

H H

H N

O

2O R1O

O

R1—CH—C—N—CH—CO2H + —CH3 + CO2 + —CH2OH

NH2H

R2

Pd

H2

—CH2—O—C—N—CH—C—N—CH—C—O—CH2—H

Phosph

N—CH—C— . . .

CH2

O

ose

PO H

3

rine

R

2

moiety

16

The sulfhydryl group of cysteine undergoes reactions typical of the –SH group. One of these reactions is the reversible oxidation with another molecule of cysteine to form the disulfide, cystine. Disulfide linkages between two cysteine residues in a polypeptide chain are a frequent occurrence. Insulin, for example, contains three disulfide linkages, two of which hold together two polypeptide chains in the physiologically active molecule. Ribonuclease similarly contains four disulfide bonds between four pairs of cysteine residues; if any one of these bonds is severed (by reduction, for example), the enzyme will lose its catalytic activity. Acid base property of amino acids. To the aqueous solutions of amino acids, Bronsted Lowry theory applies for the determination of acidic or basic nature of amino acids. The equation for the dissociation or ionization of an acid [HA] in dilute aqueous solution involves the transfer of a proton from the acid to water, which itself act as a proton acceptor to yield the acid H3O+.

HA + H2O H3O+ + A-

Here, acids that have only a weak tendency to give up proton to water are called as weak acids, while acids that readily give up their protons are termed as strong acids. The property of any given acid to dissociate is given by its dissociation constant (K);

[H3O+] [A-] K = [HA] [H2O] Here, brackets [ ] refer concentration in moles per liter. Equation can be simplified by eliminating the water molecule as;

[H+] [A-] K = [HA] The dissociation constant is based on analytically measured concentrations of reactants and products at a given total concentration and ionic strength. This constant is called apparent or concentration dissociation constant, and is designated as K’. Hence, pK' can be expressed as log of K': 1 K' pK' = log

or pK' = -log K' These pK' Values are easy to handle than K' values. Strong acids have low pK' values and strong bases have high pK' values.

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Titration of amino acids

The acid base behaviour of amino acid can be best understood in terms of Bronsted Lowry theory of acid and bases. According to Bronsted Lowry concept, an acid is a protein donor and a base is a proton acceptor. An acid base reaction always involve a conjugate acid-base pair. Proton donor Proton acceptor

CH3COOHH2PO4

- H++HPO42-

NH4+ H++NH3

H++OH-

H++ CH3COO-

HOH

For example, acetic acid (CH3COOH) is a proton donor, and acetate anion (CH3COO–) is the corresponding proton acceptor; together they constitute a conjugate acid base pair. A simple monoamino monocarboxylic α-amino acid like alanine is dibasic acid in its fully protonated form, which can donate two protons during its complete titration with a base. This two-stage titration with NaOH can be represented as:

H3NCHRCOOH + OH–

H3NCHRCOO– + OH–

H3NCHRCOO– + H2O

H3NCHRCOO– + H2O

+ +

+

The figure shows the biphasic titration curve of alanine. The pK' values of two stages of ionization of alanine yield two clearly separate legs. Each leg has a midpoint where there is minimal change in pH as OH– is added. The apparent pK' values for two stages can be determined from the midpoint of each stage; they are pK'1 = 2.34 and pK'2 = 9.69. At pH 2.34, the midpoint of the first step, equimolar concentrations of proton-donor (H3N+CHRCOOH) and proton acceptor (H3N+CHRCOO–) are present. At pH 9.69, equimolar concentration of (H3N+CHRCOO–) and H2NCHRCOO– are present. At pH 6.02, there is a point of inflection between two legs of the titration curve (Fig. 10). There is no net charge on the molecule at this pH. This is the isoelectric pH (pHI), which is the arithmatic mean of pK'1 and pK'2, that is pHI = ½ (pK'1 + pK'2). Therefore, isoelectric pH is that point at which the net charge of the molecule is zero. Titration curves of different amino acids reveals;

1. The α-carboxyl group of monoamino monocarboxylic acid is a stronger acid than the carboxyl group of comparable aliphatic acids.

2. The α-amino group of monoamino monocarboxylic acid is a stronger acid than the amino group of comparable aliphatic amines.

3. All the monoamino monocarboxylic amino acids with uncharged R groups have nearly identical pK'1 values and nearly identical pK'2 values.

4. None of monoamino monocarboxylic amino acids has significant buffering capacity at the physiologial pH zone, pH 6.0 to 8.0. They do show buffering capacity in the zones near their pK' values, i.e., pH 1.3 to 3.3 and 8.6 to 10.6.

5. The β-carboxyl group of aspartic acid and the γ-carboxyl group of glutamic acid have pK' values higher than the pK' values of the α-carboxyl groups.

6. The thiol or sulfhydryl group of cysteine and the p-hydroxyl group of tyrosine are very weakly acidic.

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7. The ε-amine group of lysine and the guanidinium group of arginine are strongly basic; they lose their protons only at a high pH. At pH 7.0, these amino acids have a net positive charge.

Fig. 10.

Separation of amino acids

The quantitative separation and estimation of amino acids is achieved by chromatographic methods. The chromatographic methods may be one of those described below; Partition chromatography

When a solute is allowed to distribute itself between equal volumes of two immiscible liquids, the ratio of the concentrations of the solute in two phases at equilibrium at a given temperature is called partition coefficient. Partition chromatography is the chromatographic separation of mixtures by the countercurrent partition coefficients by many repetitive partition steps, and this distribution is known as counter current distribution. This principle was given by LC Craig. It was first used for separation of amino acids by APJ Martin and RLM Synge. The separation is achieved in separate partition steps, which takes place on microscopic granules of a hydrated insoluble inert substance, such as silica gel. Silica gel granules are hydrophilic in nature and are surrounded by a layer of tightly bound water, which serves as a stationary aqueous phase, which flows through a moving phase of immiscible solvent containing the mixture to be separated. The mixture of solutes undergoes partition in the fixed water layer and the flowing solvent. This process occurs on the surface of each granule. The total number of partition steps in the column is so great that the different amino acids in a mixture move down the column at different rates as the moving liquid

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phase flows through it. The liquid appearing at the bottom of the column, called the eluate, is collected in the collector and analysed by ninhydrin reaction. Similarly, amino acids can be separated using filter paper chromatography. In this method, the cellulose of the filter paper fibres is hydrated. As a solvent containing an amino acid mixture ascends in a vertically held paper by capillary action, the distributions of the amino acids occur between the flowing phase and the stationary water phase bound to the paper fibres. At the end of the process, the different amino acids have moved different distances from the origin. The paper is dried, sprayed with ninhydrin solution, and heated to locate the amino acids. Ion exchange chromatography

Ion exchange chromatography was first developed by W Cohn. In this method, solute molecules are sorted by the differences in their acid base behavior. For this process a column is filled with granules of a synthetic resin containing charged resins: cationic exchangers or anionic exchangers. Amino acids are usually separated on cationic exchange columns filled with a sulfonated polystyrene resin previously equilibrated with NaOH solution in order to charge its sulfonic acid groups with Na+. This form of the resin is called the sodium form. To the washed Na+ form of resin an acid solution of the amino acid mixture is added; at pH 3.0, amino acids carry net positive charge. The cationic amino acids tend to displace the bound Na+ ions from the resin. At this pH 3.0, the most basic amino acids (Lysine, Arginine and Histidine) are bound tightly to the resin by electrostatic forces, and the most acidic amino acids (Glutamic and Aspartic acid) are bound loosely. As the pH and the NaCl concentration of eluent is gradually increased, the amino acids move down at different rates and the eluate is collected. These are analysed by ninhydrin reaction. The most anionic amino acids appear first and the most cationic amino acids appear in the last. Paper electrophoresis

Another method to separate amino acids is paper electrophoresis. In this method, a drop of a solution of the mixture is placed on a filter paper sheet moistened with a buffer of a given pH. The end of the sheet dip into electrode vessels, and an electric field is applied. Because of their different pK' values, the amino acids migrate towards either end and at different rates, depending on the pH and the electromotive force applied. eg. At pH 1.0, histidine, arginine and lysine have a charge of +2 whereas all other amino acids have a charge of +1. At pH 11.0, aspartate, glutamate, cysteine, and tyrosine have a charge of -2 and all others -1. Essential amino acids

An essential amino acid or indispensable amino acid, is an amino acid that cannot be synthesized de novo by the organism (usually referring to humans), and therefore must be supplied in the diet. Nutritional essentiality is characteristic of the species, not the nutrient. Eight amino acids are generally regarded as essential for humans. They are: isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan, and valine. In addition, the amino acids arginine and histidine are sometimes considered conditionally essential, meaning they are not normally required in the diet, but must be supplied exogenously to specific populations that do not synthesize it in adequate amounts. Patient with certain

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disease eg. Phenylketonuria (PKU) could have an additional requirement. Patients living with PKU must keep their intake of phenylalanine extremely low to prevent mental retardation and other metabolic complications. Moreover, phenylalanine is the precursor for tyrosine synthesis, a step defective in PKU, therefore phenylalanine cannot be converted to tyrosine and tyrosine becomes essential in the diet of PKU patients. The Table 2 lists the recommended daily amounts for essential amino acids in humans.

Table 2: Essential amino acids

Amino Acid Recommended Daily Allowance (RDA) mg / kg body weight

F Phenylalanine 14 L Leucine 14 M Methionine 13 K Lysine 12 I Isoleucine 10 V Valine 10 T Threonine 7 W Tryptophan 3.5 H Histidine 8 R Arginine Not known

The distinction between essential and non-essential amino acids is somewhat unclear, as some amino acids can be produced from others. The sulfur-containing amino acids, methionine and homocysteine, can be converted into each other but neither can be synthesized de novo in humans. Likewise, cysteine can be made from homocysteine but cannot be synthesized on its own. So, for convenience, sulfur-containing amino acids are sometimes considered a single pool of nutritionally equivalent amino acids. Uses of essential amino acids

Foodstuffs that lack essential amino acids are poor sources of protein equivalents, as the body tends to deaminate the amino acids obtained, converting proteins into fats and carbohydrates. Therefore, a balance of essential amino acids is necessary for a high degree of net protein utilization, which is the mass ratio of amino acids converted to proteins to amino acids supplied. Essential amino acids are synthesised by plants, microorganisms and obtained from diet. Some believe that careful monitoring of nutrient levels is important in strict vegetarian diets, but there are virtually no cases of protein-deficiency among populations consuming adequate calories. Protein-deficiency occurs among populations that are chronically undernourished. The net dietary protein utilization is profoundly affected by the limiting amino acid content (the essential amino acid found in the smallest quantity in the foodstuff), and somewhat affected by salvage of essential amino acids in the body (Table 3). The amino acids are not stored in the body. It is therefore a good idea to mix foodstuffs that will complement

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different deficiencies in their essential amino acid content. This limits the loss of nitrogen through deamination and increases overall net protein utilization.

Table 3: Illustrates sources of limiting amino acids

Protein source Limiting amino acid

Wheat lysineRice lysineMaize lysine and tryptophanPulses methionine (or cysteine) Beef phenylalanine (or tyrosine) Egg, chicken none; the reference for absorbable protein Milk or Whey, bovine methionine (or cysteine)

Mnemonics

For students wanting or needing to memorize the essential amino acids, mnemonics have been used, using the first letter of the amino acids name. The mnemonic words and phrases that have been used are, for example : PVT TIM HALL, VP MATT HILL, or THAT PILL VM. Another method uses the first letter of each essential amino acid to begin each word in a phrase, such as: "Any Help In Learning These Little Molecules Proves Truly Valuable." These include the two amino acids arginine and histidine that need some qualifications as to their requirements. Note that these devices work by using the first letter of the actual amino acids name. Due to repetition of letters, several amino acids have one-letter abbreviations that are different than their first letter (e.g. lysine has K). Thus the complete list of essential amino acids utilizing one-letter codes is MILKVWTHFR. Further, one more famous mnemonic for ten essential amino acids is “These Ten Valuable Aminoacids Have Long Preserved Life In Man” i.e, (Threonine, Tryptophan, Valine, Arginine, Histidine, Lysine, Phenylalanine, Leucine, Isoleucine, Methinine).

S. No. Feature Mnemonic Aminoacids 1 Hydrophobic aminoacids PGAVPIL Proline, Glycine, Alanine, Valine,

Phenylalanine, Isoleucine, Leucine 2 Hydroxyl group (–OH)

containing STT Serine, Tyrosine, Threonine

3 Sulphur containing CM Cysteine, Methinine 4 Basic amino acids HAL Histidine, Arginine, Lysine 5 Carboxylic group (-

COOH) containing AG Arginine and Glutamate

6 Amide group (-CONH2) containing

Ag Asparagine and Glutamine

7 With aromatic side group HTTP Histidine, Tryptophan, Tyrosine, Phenylalanine

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Water as biological solvent

Water and pH

Water makes up 60 to 90% of the weight of most forms of life. Since it is ubiquitous, thus regarded as a bland, inert liquid of living organisms. It has a higher melting point, boiling point, heat of vaporizations, heat of fusion, and surface tension than other common liquids. All these properties indicate that the forces of attraction between the molecules in liquid water and thus its internal cohesion, are relatively high. The strong intermolecular forces in water are caused by the distribution of electrons in water molecule. Each of the two hydrogen atoms shares a pair of electrons with the oxygen atom, through overlap of the 1s orbitals of hydrogen atoms with two hybridized sp3 orbitals of the oxygen atom. The H–O–H bond angle is 104.5°, and the H–O interatomic distance is 0.0965 nm. When two water molecules approach each other, electrostatic attraction occurs between the partial negative charge on the oxygen atom of one water molecule and the partial positive charge on a hydrogen atom of an adjacent water molecule. A complex electrostatic union of this kind is called as hydrogen bond. Hydrogen bonds are relatively weak as compared to covalent bond (Fig. 11).

Fig. 11: Structure of water molecule. (a) Ball and stick model; (b) Space filling model; (c). Illustrates the bond angle, hydrogen bond and covalent bond for the molecule

Water as a biological solvent

Water is a much better solvent than other common liquids. Crystalline salts, ionic compounds as well as sugar alcohols, aldehyde, ketones etc. are readily soluble in water. Water also disperses or solubilizes in the form of micelles many compounds, which contain both strongly nonpolar and strongly polar groups. Such molecules are called amphipathic eg. the sodium salt of the long chain fatty acid, oleic acid. This molecule has a single carboxyl group, which is polar and thus tends to hydrate readily, and a long hydrocarbon tail, which is nonpolar and intrinsically insoluble in water as in Fig 12. Because of this long, hydrophobic tail there is very little tendency for sodium oleate (a soap) to dissolve in water to yield a true solution. However, it readily disperses in water to form micelles, in which the negatively charged carboxylate groups are exposed and form hydrogen bonds with water

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molecules and the nonpolar, insoluble hydrocarbon chain, which do not hydrogen bond with water (Fig. 13).

CH3

O

Polar Head Non-Polar Tail

Fig. 12: Demonstration of soap micelle in water.

Fig. 13: Examples of Polar, Nonpolar and amphipathic molecules.

Body water

The body water may be divided into two main compartments: 1. Extracellular fluid (ECF), in turn consisting of plasma, interstitial fluid, and transcellular fluid (intercellular fluid); and 2. Intracellular Fluid (ICF)

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1. Extracellular fluid.

This constitutes about 30% of the total body water (TBW). In a 70 kg male with a TBW of 42 litres, 12.5 litres lie in the extracellular space. The volume of extracellular fluid is determined by the degree of dilution attained by a substance which on injection remains outside the cells. The most suitable of these is Sodium thiosulphate and the ECF as calculated by this method (termed as Thiosulphate Space) is approximately 17% of the body weight. Other diffusible substances like Inulin or Mannitol give misleading figures. (a) Plasma: This is the fluid which lies within, the heart and blood vessels and approximates 45 ml/kg body weight or 4.5% of body weight in a healthy adult. Thus, in a 70 kg man the plasma volume is about 3 litres. It is best determined by the intravenous injection of a known amount of RIHSA (Radio-iodinated human serum albumin) and working out the degree of dilution in the circulating plasma. Because of its combination with albumin the radio-iodine remains within the vascular bed and fairly accurate determinations can be made. (b) Interstitial fluid: This includes lymph and the fluid which constitutes the immediate environment of all cells in the body. It is readily obtained by subtracting the plasma volume from the volume of extracellular fluid. In a healthy adult, it is approximately 8.5 – 10 litres (21-23%) of the total body water of 42 litres. (c) Transcellular fluid: This includes fluid within the lumen of the secretory organs and anatomical spaces, viz. the gastrointestinal and biliary secretions, urine, fluid in joints and chambers of the eye, cerebrospinal fluid and fluid in the thyroid, gonads and other secretory glands. This heterogeneous but important group comprises about 2.5% of the total body water. 2. Intracellular fluid

This constitutes 70% of the TBW. Thus in a 70 kg adult with a total body water content of 42 litres, the intracellular fluid is about 30 litres. Of this 24-26 litres lies within the soft tissues (excluding fat), and about 4 litres in bone. Whereas the intracellular water of soft tissue readily participates in all fluid exchanges in the body; the water contained within bones does not do so. The intracellular fluid volume is derived by subtracting the volume of extracellular fluid from the total body water. The volumes of total body water, extracellular fluid and intracellular fluid are shown in Table 4. The metabolisms of water, Na+ and K+ are interrelated and regulated by osmotic and hormonal mechanisms in a co-ordinated manner to achieve homeostasis. The volume and osmolarity of ECF, acting through the water intake/output areas of hypothalamus, influence the secretion of anti-diuretic hormone (Arginine vasopressin, AVP) from posterior pituitary. AVP, in turn regulates water output in the kidneys. The neuro humoral mechanism, viz. the rennin-angiotensin-aldosterone system plays a crucial role in maintaining the sodium, potassium and water balance. Recently natriuretic factors, namely atrial natriuretic peptide and its various forms, and urodilatin have been implicated in the regulation of sodium balance.

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Table 4: Fluid balance in the Human Body Adult male 70 kg

(TBW is 60% of Body Weight = 42 litres)

% of Body water Volume (litres) 1. Extracellular fluid a. Plasma 7.5 3.0 b. Interstitial fluid 20.0 8.5 c. Transcellular fluid 2.5 1.0 30.0 12.5 2. Intracellular fluid 70.0 29.5 Total body water 100.0 42.0

Weak acids and bases

Acids are proton donors and bases are proton acceptors. A distinction is made, however between strong acids (eg, HCl, H2SO4), which completely dissociate into anions and cations even in strongly acidic solutions (low pH), and weak acids, which dissociate only partially in acidic solutions. A similar distinction is made between strong bases (eg, KOH, NaOH) and weak bases (eg, Ca[OH]2). Only strong bases are dissociated at high pH. Many biochemicals possess functional groups that are weak acids or bases. One or more of these functional groups- carboxyl groups, amino groups or the secondary phosphate dissociation of phosphate esters- are present in all proteins and nucleic acids, most coenzymes and most intermediary metabolites. The dissociation behavior of weakly acidic and weakly basic functional groups is therefore fundamental to understanding the influence of intracellular pH on the structure and biochemical activity of these compounds. The relative strengths of weak acids and of weak bases are expressed quantitatively as their dissociation constant. Following expression for dissociation constant (K) of two representative weak acids, R-COOH and R-NH3

+ :-

R-COOH R-COO-+ H+

[R-COO-] [H+] K = [R-COOH] R- NH3

+ R-NH2 + H+

[R-NH2] [H+] K = [R-NH3

+] Since the numeric values of K for weak acids are negative exponential numbers, it is convenient to express K as pK, where

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pK = - log K

Note that pK is related to K as pH is to H+ concentration.e.g. for a dissociation of the type :-

R- NH3+ → R- NH2 + H+

The pK refers to the pH at which the concentration of the acid, R-NH3

+, equals that of the base, R- NH2. From the above equations that relate K to [H+] and to the concentrations of undissociated acid and its conjugate base, note that when [R-COO-] = [R-COOH] or when [R-NH2] = [R-NH3

+] then K = [H+] We concluded from the above reactions that the associated (protonated) and dissociated (conjugate base) species are present in equal concentrations; the prevailing hydrogen ion concentration (H+) is numerically equal to the dissociation constant, K. If the log of both the sides of the above equation is taken and both sides are multiplied by -1, the expression would be as follows: K = [H+]

-log K = - log [H+] Since –log K is defined as pK and –log [H+] defines pH, the equation may be rewritten as: pK = pH i.e. the pK of an acid group is that pH at which the protonated and un protonated species are present at equal concentration. pH

The term pH was introduced by S.P.L. Soerensen in 1909 and is defined as the negative log of hydrogen ion concentration:

pH = -log [H+] The dissociation of water is an equilibrium process, the equation for the same is written as H2O H+ + OH-

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Thus the equilibrium constant (Keq) of water can be expressed as

[H+] [OH-] Keq =

[H2O] Here, brackets indicate concentration in moles per litre. Since the concentration of water in pure water is very high i.e, it is equal to the number of grams of H2O in a litre divided by the gram molecular weight of water, or 1000/18=55.5M and concentrations of H+ and OH-

ions are very low (1 x 10-7 M). The equilibrium constant is expressed as

55.5 Keq = [H+] [OH-] 55.5 Keq can be replaced by a constant Kw called as ion product of water.

Kw = [H+] [OH-] Value of Kw at 250 C is 1.0 x 10-14. This Kw (ion product of water) is the basis of pH scale. pH scale is a means of designating the actual concentration of H+ ions in any aqueous solution in the acidity range between 1.0 M H+ and 1.0 M OH-. The scale was devised by the Danish Biochemist, SPL Sorensen. He explained the term pH as:

[1] pH = log10

[H+]

pH = -log [H+] In a neutral solution at 250 C, [H+] = [OH-] = 1.0 x 10-7 M. Thus, pH of that solution will be,

1 pH = log

1.0 x 10-7

The higher the value of pH, the lower is the H+ concentration similarly lower the value of pH, the higher is the H+ concentration. In other words, if two solutions differ in pH by 1 unit, it means that one solution has 10 times the H+ concentration of the other. Buffers

To understand buffers, one should recall the titration curve of acid against base. Titration curve of each acid has a relatively flat zone which is about 1.0 pH unit on either side of its midpoint. In this zone, the pH of the system changes relatively little when increments of H+ or OH- are added. This is the zone in which the conjugate acid base pair acts as a buffer. At pH values outside this zone there is less capacity to resist changes in pH. The buffering power is maximum at the pH of the exact midpoint of the titration curve, at which the

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concentration of the proton acceptor is equal to that of the proton donor and pH = pK'. Buffering power of the solution decreases as the pH is raised or lowered from this point. Intracellular and extracellular fluids of living organisms contain conjugate acid–base pairs which act as buffers at normal pH of these fluids. The major intracellular buffer is the conjugate acid base pair of HPO4

--/H2PO4- [pK' = 6.8]. In continuation, Glucose-6-

phosphate and ATP impart buffering power inside the cell. The major extracellular buffer in the blood and interstitial fluid is bicarbonate buffer. The bicarbonate buffer system [H2CO3-HCO3

-] has distinctive features. It functions as a buffer in the same way as other acid-base pair, the pK' of H2CO3 is 6.1, which is far lower than the normal range of blood pH. Still it performs as physiological buffer at pH near 7.4. This is well understood by the Henry’s law of solubility of gas in water and equilibrium of carbonate ion with dissolved CO2 in blood. Blood plasma has extraordinary buffering power, which can be understood by an example. The pH of 1 litre blood plasma is altered from pH 7.4 to pH 7.2 after adding 1 ml of 10N HCl, while the same amount of HCl when added to 1 litre of normal saline, drops its pH to 2.0. Many aspects of cell structure and function are influenced by pH, the catalytic activity of enzymes is especially sensitive. All enzymes have maximal activity at a characteristic pH, called the optimum pH, and their activity declines sharply on either side of the optimum. Thus biological control of the pH of cells and body fluids is of central importance in all aspects of metabolism and cellular function. Henderson Hasselbalch equation

To understand the properties of acids and bases, titration curve is plotted for weak acids titrated with NaOH. The pH resulting after each increment of NaOH is plotted against the equivalents of OH- added. The shapes of these titration curves are similar in shape for different acids, there is slight displacement vertically along the pH scale. The pH intercept at the midpoint of the titration is numerically equal to the pK' of the acid titrated. At the midpoint, equimolar concentrations of proton donor [HA] and proton acceptor species [A-] of the acid are present. The shape of the titration curve is expressed by the Henderson-Hasselbalch equation, which is a logarithmic transformation of the expression for the dissociation constant.

[H+] [A] K' =

[HA]

[HA] H+ = K'

[A-]

calculate the negative log of both sides

[HA] -log[H+] = -log K' -log

[A-]

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since –log[H+] = pH and –log K' = pK'

[HA] pH = pK' -log

[A-]

now, the equation can be written as

[A-] pH = pK' +log

[HA] now the equation can be rewritten as

[Proton acceptor] pH = pK' +log

[Proton donor] This equation is known as Henderson-Hasselbalch equation. Using the equation, one can calculate the pK' of any acid from the molar ratio of proton donor and proton acceptor species at a given pH. Also equation helps us to calculate the pH of a conjugate acid base pair of a given pK' and a given molar ratio. However, when the concentrations of proton donor and proton acceptor are equal, the observed pH is numerically equal to the pK'. Physiological buffers

The buffer system consists of a mixture of a weak acid, HA and its salt BA, which gives the ability to the mixture to resist change in the H+ ion concentration and thus resists change in pH of the medium. Cellular metabolism predominantly yields acids. So it is appropriate that body buffers have a buffering capacity to absorb acids. The buffers are effective as long as the acid load is not excessive, and the alkali reserve is not exhausted. Once the base is utilized in this reaction, it is to be replenished to meet further challenge. Most important buffer systems of blood are as follows 1. Bicarbonate buffer system (NaHCO3/H2CO3 = [salt] / [acid]) This consists of weak acid “Carbonic acid” (H2CO3) and its corresponding salt with strong base (HCO-

3), NaHCO3 (sodium bicarbonate). This is the chief buffer of blood and the normal ratio of NaHCO3/H2CO3 in blood is 20:1. Neutralization of strong and non volatile acids entering the extracellular fluid is achieved by the bicarbonate which constitutes the so called alkali reserve. Advantages of Bicarbonate buffer system: it is efficient as compared to other system as:

• It is present in very high concentration (26-28 millimole/litre) • H2CO3, the weak acid component of buffer system is volatile and CO2 which is

exhaled out. Hence bicarbonate buffer system is directly linked up with respiration.

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2. Phosphate buffer system (Na2HPO4 / NaH2PO4 = [AlkPO4] / [acid PO4]) This consists of disodium hydrogen phosphate and sodium dihydrogen phosphate. Normal ratio in plasma is 4:1. This ratio is kept constant with the help of the kidneys. Thus, phosphate buffer system is directly linked to kidneys.

(a) When a strong acid enters the blood, it is fixed up by alkaline PO4 (Na2HPO4) which is converted to acid PO4. The acid PO4 (NaH2PO4) thus produced is excreted by the kidneys, hence urine becomes more acidic.

(b) When an alkali enters, it is buffered by the acid PO4, which is converted to alkaline PO4 and is excreted in urine producing increased alkalinity of urine.

Thus phosphate buffer system works in conjunction with the kidneys. A normal healthy kidney is necessary for proper functioning. 3. Protein buffer system (Na+ Pr- / H+ Pr- = [Salt] / [Acid])

• In acidic medium: Protein acts as base and takes up H+ ions from the medium forming -NH3

+ and –COOH and proteins become positively charged. • In alkaline medium: Protein acts as an acid gives H+, forming COO- and –NH2

groups. H+ combines with OH- to produce a molecule of water and proteins become negatively charged.

4. Hemoglobin buffer System: The buffering capacity of hemoglobin, as of any protein, depends on the number of dissociable buffering groups viz., acidic COOH group and basic-NH2 group, Guanidino group and most important is imidazole group, which varies with the pH of the medium. With the pH range of 7.0 to 7.8, most of the physiological buffering action of Hb is due to the “imidazole” group of amino acid “histidine”. Haemoglobin contains two groups:

• Fe++ containing group which is concerned with carriage of O2 and • Imidazole N2 group (pK = 6.7), which can give up H+ (proton) and accept H+

depending on the pH of the physiological medium. Thus, buffering capacity of Hb is due to the presence of imidazole nitrogen group which remains dissociated in acidic medium and conjugate base forms.

Oxygenated Hb is a stronger acid than deoxygenated Hb. On oxygenation, the imidazole N2 group acts as stronger acid and protons dissociate into the medium.

Deoxygenated Hb is less acidic, less dissociable and imidazole N2 group acts as conjugate base and takes up protons from the medium.

Acidity of the medium therefore favors delivery of O2 Alkalinity of the medium similarly favors oxygenation of Hb.

Fitness of the aqueous environment for living organisms

Water constitutes a principal end product of oxidative metabolism of foods and it is the solvent for all processes in the human body. It transports nutrients to cells, determines cell volume by its transport into and out of cells, removes waste products via urine, and acts as the body’s coolant via sweating. An excellent nucleophile, water serves as a reactant in- and

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as a product of-many metabolic reaction. The active sites of enzymes are constructed so as to either exclude or include water depending on whether water is or is not a reactant. Homeostasis, the maintenance of the composition of the internal environment that is essential for health, includes consideration of the distribution of water in the body and the maintenance of appropriate pH and electrolyte concentration. Regulation of water balance depends on hypothalamic mechanisms for controlling thirst, on antidiuretic hormone, and on retention or excretion of water by kidneys and evaporative losses due to respiration and perspiration. Maintenance of the extracellular fluid between pH 7.35 and 7.45, in which the bicarbonate buffer system plays a key role, is essential for health. Disturbances of acid-base balance causes acidosis (blood pH < 7.35) and alkalosis (blood pH>7.45). Hydrogen bonds. Water molecules have ability to form hydrogen bonds and this also account for its ability to dissolve many organic molecules. Macromolecules such as proteins and nucleic acids that are stabilized by intermolecular hydrogen bonds may exchange surface hydrogen bonds for hydrogen bonds to water, enhancing solubility. Water modifies the properties of biomolecules such as proteins and amino acids that possess both polar and nonpolar functional groups. Entropic forces dictate that macromolecules in aqueous solutions fold such that their polar portions contact the water interface while their nonpolar portions are buried in the interior of the biomolecules Electrostatic, hydrophobic interactions and vander Waals forces also play vital roles in maintaining molecular structure, including the structure of proteins. Excellent nucleophile: Many metabolic reactions involve attack by electron rich molecules or ions known as nucleophiles (“nuclus seekers”).Common intracellular nucleophiles include the oxygen of water, the oxygen of inorganic and organic phosphates, the sulphur of thiols and the nitrogen of amines. Reactions that biosynthesize or degrade proteins, nucleic acids and lipids all involve attack by nucleophiles. The release of glucosyl units from the glycogen serves in muscle tissue as an energy reserve and in liver to maintain blood glucose levels. Enzyme catalysed nucleophilic attack by oxygen of inorganic orthophosphate on the 1, 4-glycosidic bonds of glycogen releases glucose-1-phosphate. Similarly rupture of the 1, 6-glycosidic bonds involves nucleophilic attack by the oxygen atom of water. Heat buffer: The high specific heat of water is useful to large terrestrial animals, because body water acts as a heat buffer, allowing the body temperature to stay constant despite temperature fluctuations. Secondly, the high heat of evaporation of water is very helpful to vertebrates as an effective means of losing heat by evaporation of sweat. Also the high degree of internal cohesion of water enables higher plants to transport dissolved nutrients from roots to leaves via transpiration. All these properties make water a unique aqueous phase for survival apart from being a solvent.

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Suggested Reading 1. Lehninger’s Principle of Biochemistry by A H Lehninger, D L Nelson and M M Cox. Fourth edition, W

H Freeman & Company Ltd, New York, 2004. 2. Biochemistry by Lubert Stryer, J M Berg and J L Tymoczko. Fifth edition, W H Freeman & Company

Ltd, New York, 2002.

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