Abstract
We tried to develop an estimation method for γ-carboxyglutamic acid in a protein. A proposed analytical method is as follow. Hydrolysis of a peptide or a protein {(0.51.0)mg} was performed with 0.1ml of 2.5 N NaOH at 110°C for four to twenty four hrs. The hydrolyzate is neutralized to pH 3.25 with 0.26ml of 1 N HCl and lyophilized. The lyophylized sample was dissolved in 1.5ml of 0.1 N Na-citrate buffer (pH 3.25). 0.5ml of the sample solution was analyzed by an automatic amino acid analyzer (with acidneutral column). γ-Carboxylglutamic acid was eluted at the interval of (20±0.5)min after the introduction of the sample, where aspartic acid was eluted at 46min site. The color value was 0.39 of that of glutamic acid. The overall recovery was (98±3)%. The method was tested with several γ-carboxyglutamic acid containing peptides. It is known that there are γ-carboxylglutamic acid in prothrombin and all of them are localized in the N-terminal part of the protein and none are in the C-terminal part (thrombin). From the application results of the present method, about ten residues of γ-carboxyglutamic acid was found in prothrombin as well as in N-terminal peptides, prepared by two different procedures and none were found in thrombin and also in serum albumin, which was used as a control substance.
