A fluorescence study of DNS-labelled chymotrypsin and related enzymes

Abstract

Various dansyl (DNS)-conjugates of chymotrypsin, chymotrypsinogen and elastase have been prepared. Excitation and emission spectra, and fluorescence depolarization measurements have been obtained in a spectrophotofluorometer, and relevant fluorescent lifetimes have been derived from fluorescence decay measurements. Free DNS-OH gave a lifetime of 13 nsec in fair agreement with other estimates, but combined DNS gave significantly lower values. In particular, ϵ-DNS lysine in water gave a value of 4 nsec, with higher values (up to 10 nsec) at increasing glycerol concentration. Of the various protein conjugates, specifically labelled chymotrypsin gave the lowest lifetime (6 nsec); for the others, higher values were observed, the values increasing with pH of labelling. Of the protein conjugates, only in the case of specifically labelled elastase, did 'glycerol concentration cause an increase in the measured τ value from 8 to 12 nsec. Active-site labelled chymotrypsin was unusual in that its fluorescent spectrum was dependent upon excitation wavelength and its excitation spectrum upon the selected emission wavelength. The addition of further non-specifically attached DNS-groups caused a shift towards the spectra of such DNS-groups, and saturation with indole gave spectra of such a type as well as erasing wavelength dependence. It seemed that specifically attached DNS groups were probably within the “tosyl hole” but could be dislodged by indole, a competitive inhibitor. Depolarization properties were in all cases markedly dependent upon excitation wavelength. Experiments performed with varying temperature gave relaxation times which were smaller than expected for a rigid molecule with the molecular weight of chymotrypsin but, also, considerable increases were observed with increasing excitation wavelength. It seemed that the susceptibility to temperature-activated rotation of the DNS group increased with the energy of the incident radiation. At constant temperature, apart from specifically-labelled chymotrypsin, spuriously high relaxation times were observed, for which an explanation is required. Until this is provided, depolarization measurements cannot be regarded as providing a sound method of measuring relaxation times of protein molecules in solution.