Complex formation of guanidinated bovine trypsin inhibitor (Kunitz) with trypsin, chymotrypsin and trypsinogen as studied by the spin-label technique.

2.1. Materials One approach to circumvent this difficulty is to introduce an indirect signal by coupling an indicator reaction to the reaction of interest. Examples are monitoring the enzymatic activity of free enzyme [2] or following the displacement of proflavin from the enzyme when the inhibitor is bound [3]. With all these indirect measurements careful studies as to possible side reactions and influences on the equilibrium and kinetic properties of the enzyme/inhibitor system have to be included. Another approach is to covalently attach reporter groups to the reaction partners. The association of chymotrypsin and the bovine trypsin inhibitor (Kunitz) for example was studied using covalently bound fluorescent labels [4]. Employing these techniques always comprises to show that the reporter groups are tolerable perturbations of the system under investigation. Bovine trypsin (EC 3.4.21.4), chymotrypsin (EC 3.4.21 .l) and trypsinogen were obtained from Merck (Darmstadt). 3.Maleimido-2,2,5,5_tetramethylpyrrolidine-I -oxyl was purchased from Syva (Palo Alto CA). The bovine trypsin inhibitor (Kunitz), Trasylol@, was kindly provided by Bayer (Wuppertal).