alpha-chymotrypsin and rhodamine-110

Single molecule fluorescence spectroscopy offers great potential for studying enzyme kinetics. A number of fluorescence reporter systems allow for monitoring the sequence of individual reaction events with a confocal microscope. When using a time-correlated single photon counting (TCSPC) detection scheme, additional information about the fluorescence lifetimes of the fluorophores can be obtained. We have applied a TCSPC detection scheme for studying the kinetics of α-chymotrypsin hydrolyzing a double-substituted rhodamine 110-based fluorogenic substrate in a two-step reaction. On the basis of the lifetime information, it was possible to discriminate the intermediate and the final product. At the high substrate concentration used, only the formation of the intermediate was observed. No rebinding of the intermediate followed by rhodamine 110 formation occurred at these high concentrations. We have further found no alterations in the fluorescence lifetime of this intermediate that would indicate changes in the local environment of the fluorophore originating from strong interactions with the enzyme. Our results clearly show the power of using lifetime-resolved measurements for investigating enzymatic reactions at the single molecule level.

Topics: Animals; Biocatalysis; Cattle; Chymotrypsin; Kinetics; Models, Molecular; Protein Conformation; Rhodamines; Rotation; Spectrometry, Fluorescence; Time Factors

Commonly used fluorogenic substrate analogues for the detection of protease activity contain two enzyme-cleavable bonds conjugated to the fluorophore. Enzymatic cleavage follows a two-step reaction with a monoamide intermediate. This intermediate shows fluorescence at the same wavelength as the final product complicating the kinetic analysis of fluorescence-based assays. Fluorogenic substrate analogues for α-chymotrypsin with one cleavable peptide bond have been prepared from morpholinecarbonyl-Rhodamine 110 (MC-Rh110). A comparison of their kinetic properties with the corresponding (peptide)(2)-Rh110 derivatives revealed that these frequently used double-substituted substrate analogues yield only apparent K(m) and k(cat) values that are quite different from the kinetic parameters obtained from the monosubstituted MC-Rh110 based substrate analogues. Although both the monoamide intermediate and MC-Rh110 are monosubstituted Rhodamine 110 derivatives, they show different spectroscopic properties. The data from the spectroscopic analysis clearly show that these properties are directly related to the electron structure of the fluorophore and not to the previously proposed equilibrium between the lactone form and the open ionic form of the fluorophore. This knowledge about the determinants of the spectroscopic properties of monosubstituted Rhodamine 110 introduces a way for a more systematic development of new fluorogenic protease substrate analogues.

Topics: Animals; Cattle; Chymotrypsin; Fluorescent Dyes; Kinetics; Models, Molecular; Morpholines; Oligopeptides; Pancreas; Rhodamines; Spectrometry, Fluorescence; Substrate Specificity