A micromethod for the determination of N- and O-acetyl groups.

In connexion with studies on the N-terminal groups of histones (Phillips, 1961), a method was needed for measuring about 0-1 ,umole of aliphatic acyl groups derived from a few milligrams of protein. Initial experiments were done in which acetylamino acids and ovalbumin were hydrolysed in sulphuric acid and an ether extract was chromatographed, by using modifications of the method of Brown & Hall (1950). The acetate spot was readily detectable by indicators but it was transient and the method could not be made quantitative. When the extract was treated with hydrazine, acetic acid hydrazide could be demonstrated (Satake & Seki, 1950), but it could not be measured and, moreover, in the hydrazinolysate of a protein, the large amounts of amino acid hydrazides hinder the detection of unaminated hydrazides. The reaction of amino acid hydrazides with 1-fluoro-2,4-dinitrobenzene has been used in a method of analysis of C-terminal groups of proteins (Ohno, 1953), and it was considered that the formation of aliphatic acid DNP-hydrazides (R CO NH*NH-DNP) would allow their measurement provided that they could be separated from the very large amounts of DNPamino acid hydrazides formed concomitantly. DNP-hydrazides have been used for the qualitative analysis of the aliphatic acid components of natural fats and oils (Inouye & Noda, 1955), though in this case the derivatives were formed by reaction of the acid chlorides with 2,4-dinitrophenylhydrazine, a procedure which could probably not be applied to proteins.