CONVENIENT MICROMETHOD FOR THE ASSAY OF PRIMARY AMINES AND PROTEINS WITH FLUORESCAMINE - RE-EXAMINATION OF THE CONDITIONS OF REACTION

Fluorescamine, a reagent capable of reacting with a wide variety of nucleophiles, only gives fluorescent products when reacted with primary amines. The results of a reexamination of the principal parameters affecting the reaction are presented with emphasis on practical aspects. In particular, the potential competition of other nucleophiles (secondary amines and thiols) and the effect of the pH on the reaction and on the fluorescence of the product are presented. Polyalcohols, due to their reversible interaction with fluorescamine, affect the velocity without interfering with its extent. The initial hydrolysis product of fluorescamine under mild alkaline conditions has been isolated and characterized. Although unreactive with most amines, it is shown that at pHs below 7 it will react with amines that show abnormal low pH-dependent reactivity with fluorescamine. Histidine is the only amino-acid that under these conditions will react giving a fluorescent product. Among other amino groups containing molecules tested, only polyamines, histamine, and N-amino terminal-free peptides have been found to give this type of reaction. The factors affecting the limit of sensitivity of the reaction are also studied. The very low, but significant, fluorescence of the blanks sets the actual limit of sensitivity of the reaction. A micromethod that reduces the amount of fluorescamine in the assay under conditions in which the reaction is quantitative is described. A similar micromethod can be used with the O-phtalaldehyde reaction. The particular application of fluorescamine in the assay of proteins is compared to other methods. Alkaline and acid hydrolysis are shown to decrease the variability of the reaction shown by different proteins, while increasing the sensitivity. The different practical alternatives in the assay of proteins with fluorescamine and O-phtalaldehyde are discussed. © 1979 Academic Press, Inc.