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 alkylatio n 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 h ydrolysis of the alkylated protein, of the residual lysine. When the n ative proteins were analyzed, only 50% of NH2 groups were quantified b y TNBS, 80% by OPA and modified OPA, while the recovery in lysine was slightly higher than 100% by amino acid analysis. The highest repeatab ility was obtained with the OPA methods. For the measurement of alkyla tion degrees, those obtained by the TNBS procedure were more variable than by the OPA methods. Moreover, the TNBS procedure is rather long a nd tedious. The modified OPA method (involving N, N dimethyl-2-mercapt oethylammonium chloride instead of 2-mercaptoethanol) gives approximat ely the same results as OPA method. It has the advantage to be more co nvenient, since the absorbance of the isoindoles obtained is stable fo r at least one hour. The amino acid analysis enables to quantify the l oss in lysine, and possibly to identity the alkyl-lysine. However colo rimetric methods are shorter and less tedious.