The thiol group is of great importance in various fields of biological sciences. Thiol-cont,aining substances of biological interest include proteins (1) and low molecular compounds (2) such as cysteine, reduced glutathione, ergothionine, and some coenzymes. The available methods of assay for these thiol compounds consist of the classic nitroprusside method (3) and of iodometric (4)) amperometric ( 5)) spectra-photometric (6)) and calorimetric (7) titrations.
These methods require a sample of at least several milliliters containing about 1O-5 M of a thiol compound. Such amounts are not always available and there is obviously an urgent need for a more sensitive method of analysis in the fileds of biological and medical sciences.
We have reported (8,9) that certain nonfluorescent N-substituted maleimides react with thiol compounds to give addition products, which are strongly fluorescent. The reaction of N-(p-(2-benzimidazoly) phenyl)maleimide (see I in Scheme 1) (9) with simple thiol compounds such as /3-mercaptoethanol and reduced glutathione has now been examined to determine the general aspects of these reactions with emphasis on fluorescent characteristics. The reaction was also applied to fluorometry of these low molecular thiol substrates with the aim of determining practical experimental conditions for the microdetermination of such compounds in biological samples.