Abstract
SUMMARY Ribonuclease is inactivated by iodoacetate at 40”, the rate depending upon the pH at which reaction is carried out. It, is most rapid between pH 5.5 and 6, and at, pH 2.8, less rapid at intermediate pH values and under more alkaline conditions (pH 8.5 to 10). The variation in rate is a consequence of the fact that different types of amino acid residues in the ribonuclease molecule react with iodoacetate at the different pH values. Amino acid analyses showed that at pH 5.5 to 6, a histidine residue is involved, at pH 8.5 to pH 10 reaction occurs with the e-amino groups of lysine residues, whereas at pH 2.8, the thio- ether sulfur of one or more methionine residues is alkyla,ted. After removal of iodoacetate, the various reaction mixtures were chromatographed on columns of IRC-50 in an attempt to isolate purified carboxymethylated derivatives. From the mixture present after reaction at pH 5.5, it was possible to isolate a chromatographically homogeneous protein that was totally in- active and differed from ribonuclease A only in that the imidaz- ole group of a single histidine residue had been carboxymethyl- ated. The fact, that the pH optimum for the reaction leading to the formation of imidazole carboxymethyl ribonuclease is around pH 5.5 to 6, instead of at the more alkaline pH values observed for the alkylation of cy-N-acetylhistidine, suggests that inactivation takes place as a result of substitution on a particular one of the four histidine residues present in ribonuclease.
Highlights
Amino AcidT This corrected value for half-cystine is high probably because the amount of decomposition on hydrolysis was less than usual
Reaction with Histidine Residues at pH 5.5-The effluent curve obtained from a hydrolysate of a sample of ribonuclease A about half-inactivated at pH 5.5 showed a small new peak at 180 ml., just ahead of and partly overlapping proline. (The analysis shown in Fig. 2b is for the chromatogrnphically purified derivative isolated from the heterogeneous reaction mixture in the manner described later.) The material in this new peak reacts with ninhydrin to give a substance absorbing maximally at 570 ml*, so that it is clearly distinguishable from proline, which gives a product with ninhydrin that absorbs at 440 rnp
At neutral or slightly acid pH values, the most rapid reaction seems to be with the imidazole group of a histidine residue, but longer contact between the reactants will lead to coverage of other groups, notably the sulfur atoms of methionine residues [20], and the e-amino groups of lysine residues as well
Summary
T This corrected value for half-cystine is high probably because the amount of decomposition on hydrolysis was less than usual. The uncorrected value is 7.6 residues per molecule. $ Figures in boldface type differ significantly from the values normally obtained with untreated ribonuclease A
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