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An isotopic method for determining chemically reactive lysine based on succinylation
Author(s) -
Anderson Trevor R.,
Quicke George V.
Publication year - 1984
Publication title -
journal of the science of food and agriculture
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.782
H-Index - 142
eISSN - 1097-0010
pISSN - 0022-5142
DOI - 10.1002/jsfa.2740350418
Subject(s) - succinylation , succinic anhydride , lysine , chemistry , chromatography , trichloroacetic acid , lysozyme , acetic anhydride , bovine serum albumin , guanidine , hydrochloride , acetylation , carbodiimide , biochemistry , amino acid , organic chemistry , gene , catalysis
A method is described for the routine succinylation of proteins using [ 14 C]succinic anhydride as a means of measuring free ϵ‐amino groups. Maximal succinylation was achieved using 6 M guanidine hydrochloride as protein solvent and an 80‐fold molar excess of succinic anhydride relative to total lysine residues. Treatment with hydroxylamine (pH 13, 25°C, 5 min) removed unwanted O‐succinyl esters. Succinylated protein was precipitated with trichloroacetic acid, residual label washed out with ethanol and the extent of labelling measured in a scintillation counter. The method gave close to theoretical values (n.s., P >0.05) for lysyl residues in egg white lysozyme, bovine haemoglobin, ovalbumin and bovine serum albumin but gave a low value with β‐lactoglobulin and an overestimate with insulin attributable to the relatively high contribution of α‐ relative to ϵ‐amino groups in this low molecular weight protein. The method gave good results for 12 soya protein samples and was shown to be very sensitive to ‘isopeptide‐type’ heat damage in these samples. The results correlated well ( r = 0.91) with those obtained by the well established dye‐bound lysine difference procedure whereas, as could be expected, both these methods correlated poorly with total lysine determinations ( r =0.69 for succinic anhydride and r =0.77 for the dye‐binding method). The proposed method shows promise as a rapid procedure for the estimation of available lysine, but further studies are necessary to test its ability to measure nutritionally available lysine in all categories of heat damage.