States of Amino Acid Residues in Proteins: XXIV. Fluorescence of 3-Benzyl-4-methyl-7-diethylaminocouniarin in the Presence of α-Chymotrypsin and Its Derivatives

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Abstract
The quantum yield and the maximum wavenumber of the fluorescence of a coumarin dye, 3-benzyl-4-methyl-7-diethylaminocoumarin (BMDC) were measured in water in the presence and absence of α-chymotrypsin [EC 3.4.4.5] or its inactive forms such as chymotrypsinogen, alkali-denatured α-chymotrypsin, N-tosylphenylalanyl chloromethyl ketone (TPCK)-modified chymotrypsin and diisopropylfluorophosphate (DIP)-modified chymotrypsin. A great increase of the quantum yield of fluorescence and a blue shift of the fluorescence band were found by addition of α-chymotrypsin while addition of chymotrypsinogen, TPCK- and DIP-chymotrypsin caused only small changes of the fluorescence band. The changes caused by α-chymotrypsin were suppressed by co-existence of β-phenylpropionic acid, a competitive inhibitor of the enzyme. The fluorescence of 4-methyl-7-diethylaminocoumarin with no benzyl group was less affected by α-chymotrypsin. The fluorescence band of BMDC was enhanced and shifted similarly when the medium was changed from water to alcohols or ethanol-water mixtures. It was deduced from these results that BMDC is adsorbed on the specificity-determining site of α-chymotrypsin which is to be of aromatic nature and that the site is formed on activation of chymotrypsinogen.

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