DETERMINATION OF ALKYLATION DEGREE BY 3 COLORIMETRIC METHODS AND AMINO-ACID-ANALYSIS - A COMPARATIVE-STUDY

Beta-casein and beta-lactoglobulin have been alkylated by acetaldehyde to different degrees by varying the aldehyde/NH2 ratio. The alkylation rate was determined either by quantitative analysis of amino groups not involved in the alkylation process by trinitrobenzenesulfonic acid (TNBS) or o-phthaldialdehyde (OPA), or by determination, after acid hydrolysis of the alkylated protein, of the residual lysine. When the native proteins were analyzed, only 50% of NH2 groups were quantified by TNBS, 80% by OPA and modified OPA, while the recovery in lysine was slightly higher than 100% by amino acid analysis. The highest repeatability was obtained with the OPA methods. For the measurement of alkylation degrees, those obtained by the TNBS procedure were more variable than by the OPA methods. Moreover, the TNBS procedure is rather long and tedious. The modified OPA method (involving N, N dimethyl-2-mercaptoethylammonium chloride instead of 2-mercaptoethanol) gives approximately the same results as OPA method. It has the advantage to be more convenient, since the absorbance of the isoindoles obtained is stable for at least one hour. The amino acid analysis enables to quantify the loss in lysine, and possibly to identity the alkyl-lysine. However colorimetric methods are shorter and less tedious.