An isotopic method for determining chemically reactive lysine based on succinylation

Abstract

A method is described for the routine succinylation of proteins using [i4C]succinic anhydride as a means of measuring free .+amino groups. Maximal succinylation was achieved using 6~ 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 0-succinyl esters. Succinylated protein was precipitated with tnchloroacetic 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 /3-lactoglobulin and an overestimate with insulin attributable to the relatively high contribution of a-relative to &-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.

Non-Enzymatic Degradation Pathways of Lactose and Their Significance in Dairy Products

“…14 C]succinic anhydride (Anderson and Quicke, 1984) and fluorodinitrobenzene (FDNB; Carpenter, 1960;Mottu and Mauron, 1967). Although still used occasionally to measure the destruction of lysine in food systems, the TNBS method is very unsatisfactory for this purpose due to reaction of the reagent with the Amadori adduct (Hurrell and Carpenter, 1974).…”

Advanced Dairy Chemistry Volume 3

“…Amadori adducts). Several methods are available for the chemical estimation of reactive E-NH2 groups in proteins, including reaction with trinitrobenzene sulphonic acid (TNBS; Kakade and Liener, 1969), o-methylisourea (Mauron and Bujard, 1964), borohydride (Hurrell and Carpenter, 1974), [ 14 C]succinic anhydride (Anderson and Quicke, 1984) and fluorodinitrobenzene (FDNB;Carpenter, 1960;Mottu and Mauron, 1967). Although still used occasionally to measure the destruction of lysine in food systems, the TNBS method is very unsatisfactory for this purpose due to reaction of the reagent with the Amadori adduct (Hurrell and Carpenter, 1974).…”