Influence of various fluorescent and non-fluorescent labels on the kinetics of the complex formation of alpha-chymotrypsin with basic pancreatic trypsin inhibitor (Kunitz).
The association of alpha-chymotrypsin with basic pancreatic trypsin inhibitor was studied using extrinsic signals produced by fluorescent and nonfluorescent labels. The reactive dyes were covalently bound to the proteins in the complexed state, in which the binding region was protected. The signals were sufficiently large to measure the complex formation at protein concentrations of 10(-9)M by fluorescence and down to 10(-6)M by absorption. Therefore, the association and dissociation could be followed over a broad range of concentration. Good correspondence was observed between data which were obtained with different labels and with published values for the unlabeled proteins. Existing differences could be explained by different buffer conditions used by the different authors. Also the pH dependence of the dissociation rate constants was essentially unaltered by the introduction of the labels. The large signals allowed a direct measurement of the equilibrium constants of dissociation, even at high pH, at which they are in the range of 10(-8)M. The experimentally determined binding constants were in agreement with those calculated from the rate constants. The temperature dependence of the binding constants revealed a small positive and pH-dependent enthalpy change [deltaHo = 4.0 kcal/mol (16.7 kJ) at H 7.0[. The results prove that the labeling can be performed in such a way that the equilibrium and kinetic parameters of the system studied are not significantly influenced.
D000345:Affinity Labels / Q000494:pharmacology*; D000818:Animals; D007611:Aprotinin / Q000378:metabolism*; D001391:Azo Compounds / Q000378:metabolism / Q000494:pharmacology; D002021:Buffers; D002417:Cattle; D002918:Chymotrypsin / Q000378:metabolism*; D003619:Dansyl Compounds / Q000378:metabolism / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000494:pharmacology*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D011485:Protein Binding; D013696:Temperature
D000345:Affinity Labels; D001391:Azo Compounds; D002021:Buffers; D003619:Dansyl Compounds; D005456:Fluorescent Dyes; D007611:Aprotinin; D002918:Chymotrypsin