Arginine ionization

The most significant amino acids for modification and conjugation purposes are the ones containing ionizable side chains aspartic acid, glutamic acid, lysine, arginine, cysteine, histidine, and tyrosine (Figure 1.6). In their unprotonated state, each of these side chains can be potent nucleophiles to engage in addition reactions (see the discussion on nucleophilicity below). 

In the case of carboxypeptidase B, Shaklai et al.(2lT> compared the relative contributions to the protein phosphorescence from tyrosine and tryptophan for the apoenzyme, the zinc-containing metalloenzyme in the absence of substrate, the metalloenzyme in the presence of the substrate iV-acetyl-L-arginine, and the metalloenzyme in the presence of the specific inhibitor L-arginine. The tyrosine tryptophan emission ratio of the metalloenzyme was about a factor of four smaller than that of the apoenzyme. Binding of either the substrate or the inhibitor led to an increase in the emission ratio to a value similar to that of the apoenzyme. The change in the tyrosine tryptophan phosphorescence ratio was attributed to an interaction between a tyrosine and the catalytically essential zinc. The emission ratio was also studied as a function of pH. The titration data are difficult to interpret, however, because a Tris buffer was used and the ionization of Tris is strongly temperature dependent. In general, the use of Tris buffers for phosphorescence studies should be avoided. 

Some amino acids have additional ionizable groups in their side-chains. These may be acidic or potentially acidic (aspartic acid, glutamic acid, tyrosine, cysteine), or basic (lysine, arginine, histidine). We use the term potentially acidic to describe the phenol and thiol groups of tyrosine and cysteine respectively under physiological conditions, these groups are unlikely to be ionized. It is relatively easy to calculate the amount of ionization at a particular pH, and to justify that latter statement. 

Similar calculations as above for the basic side-chain groups of arginine pK 12.48) and lysine pK 10.52), and the acidic side-chains of aspartic acid (pATa 3.65) and glutamic acid (pAfa 4.25) show essentially complete ionization at pH 7.0. However, for cysteine (pATa of the thiol group 10.29) and for tyrosine (pAfa of the phenol group 10.06) there will be negligible ionization at pH 7.0. 

When the R group contains another ionizable group, the amino acid will have more than two dissociation constants. The carboxylic acid gronps of aspartic acid and glutamic acid, the amine of lysine, and the guanidino group of arginine will all... 

The amino acids in question are the basic amino acids lysine, arginine, and histidine, and the acidic amino acids aspartic acid and glutamic acid. The side-chain functions of these amino acids, ionized at pH 7 (see Box 4.7), act as acids or bases. In a reverse sequence, protons may be acquired or donated to regenerate the conjugate acids and conjugate bases. 

We have shown the cysteine thiol group as uncharged. The pAfa for this group in cysteine is about 10.3, and application of the Henderson-Hasselbach equation (see Section 4.9) indicates there will be negligible ionization at pH 7. Nevertheless, under the influence of a suitable basic group, e.g. arginine pATa 12.5, ionization to thiolate may be possible. In such an environment, thiolate may act as the nucleophile in the mechanism. 

The exact ionic state of the side chains in the last two classes will depend on the pH of the solution. At pH 7.0 the side chains of glutamate and aspartate have ionized carboxylates, and the side chains of lysine and arginine have positively... 

The side chains of the basic amino acids accept protons (see Hgure 1.3). At physiologic pH the side chains of lysine and arginine are fully ionized and positively charged. In contrast, histidine is weakly basic,... 

Figure 4-18 (A) Stereoscopic view of the interactions between the E. coli galactose chemoreceptor protein and a hound molecule of D-glucose. (B) Schematic drawing showing many of these interactions and the state of ionization deduced for the aspartate and arginine side chains. From Vyas et al.m...

An additional point should be noted from table 3.3. Whereas the amino acid side chains (R groups) that are normally charged at physiological pH are restricted to five amino acids (aspartic acid, glutamic acid, lysine, arginine, and sometimes histidine), a number of potentially ionizable R groups are part of other amino acids. These include cysteine, serine, threonine, and tyrosine. The ionization reac-... 

Titration curve of /3-lactoglobulin. At very low values of pH (<2) all ionizable groups are protonated. At a pH of about 7.2 (indicated by horizontal bar) 51 groups (mostly the glutamic and aspartic amino acids and some of the histidines) have lost their protons. At pH 12 most of the remaining ionizable groups (mostly lysine and arginine amino acids and some histidines) have lost their protons as well. 

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