Quantitation of methionyl peptides in nanomole quantities by a fluorometric method

A quantitative and highly specific method to determine low concentrations of methionyl peptides, which do not contain tryptophan or cysteine residues, has been developed. The method is based on the stoichiometry and selectivity of N-chlorosuccinimide (NCS) towards methionine and N-acetyltryptophan. N-Chlorosuccinimide reacts with N-acetyltryptophan in a 1:1 ratio to produce the N-acetyl-2-oxindolealanine--a derivative essentially devoid of fluorescence. The decrease in fluorescence intensity is approximately linear with respect to the NCS concentration. Preincubation of NCS with methionine or methionyl peptide consumes a stoichiometric amount of the reagent and the unreacted NCS is quantitated by the decrease in fluorescence intensity resulting upon incubation of the mixture with 1 eq of N-acetyltryptophan. Less than 1 nmol of methionyl peptide can be accurately quantitated by this method.