Acetylation of Tyrosine in Pepsin

Abstract

Crystalline 60 per cent active acetyl pepsin has 7 acetyl groups per mol of pepsin, 3 of which are readily hydrolyzed in acid at pH 0.0 or in weak alkali at pH 10.0. The tyrosine-tryptophane content of this acetylated pepsin, measured colorimetrically, is less than pepsin by three tyrosine equivalents. Hydrolysis at pH 0.0 or pH 10.0 of the 3 acetyl groups results in a concomitant increase in the number of tyrosine equivalents. In the pH 0.0 hydrolysis experiment there is also a simultaneous increase in specific activity. The phenol group of glycyl tyrosine is acetylated by ketene under the conditions used in the acetylation of pepsin and the effect of pH on the rate of acetylation is similar in the two cases. It is concluded that the acetyl groups in the 60 per cent active acetyl pepsin, which are responsible for the decrease in specific enzymatic activity, are 3 in number and are attached to 3 tyrosine phenol groups of the pepsin molecule.

N-Acetyl Groups in Wool and Extracted Wool Proteins

“…Although Herriott (1935) found that alkaline hydrolysis was satisfactory for the' determination of acetyl groups in acetylated proteins, our experiments show that acid hydrolysis followed by steam distillation is the preferable procedure, at least for proteins such as wool with a relatively high sulphur content, and very low acetyl content.…”

“…(i) Total Acetyl Groups.-The method recommended by Herriott (1935) for the determination of N-acetyl groups in acetylated proteins was found to be unreliable for the proteins studied here. This seems to be due at least in part to volatile acids produced by the alkaline degradation of sulphur-containing amino acids which are present in large amounts in comparison with the small quantity of acetyl groups.…”

“…They recovered the acetic acid by vacuum distillation under conditions similar to those used by Herriott (1935) but reported that blank values for untreated proteins were in the range 1-3 equivalents per 10 4 g. As previously mentioned we were unable to satisfactorily distil sulphuric acid solutions under these conditions. However, the steam-distillation procedure gave low and reproducible blank values and N-acetyl methionine gave 99% recovery of acetyl groups after hydrolysis with acid.…”

“…Initially, the procedure recommended by Herriott (1935) for the determination of acetyl groups in acetylated proteins was used. This involved hydrolysis of the protein (0·5 g) for 5 days at 37°C in 0·35N alkali followed by repeated vacuum distillation at 50±5°C of the solution acidified with aqueous citric acid.…”

“…(ii) O-Acetyl Group8.-0-acetyl determinations on wool were carried out as described by Herriott (1935) and the alkaline liquor distilled after acidification with citric acid.…”