HEPARIN-INDUCED STRUCTURAL AND FUNCTIONAL ALTERATIONS OF BOVINE TRYPSIN

To investigate the mechanism whereby heparin can modulate the activity of serine proteinases, bovine trypsin was chosen as reference and tre ated with heparin at 10, 100 and 200 mu g/ml, in buffer solvents, with and without incubation at 37 degrees C. Heparin caused rapid, buffer- and pH-dependent decrease in trypsin solubility due to the generation of insoluble fragments from proteinase. Desalting treatments variousl y restored solubility by removing insoluble material. UV absorption an d fluorescence emission spectra revealed significant heparin-induced c onformational alterations in the trypsin molecule, the maximal effect being apparent at a proteinase-to-heparin molar ratio ranging from 1.6 to 1.0. The involvement of the catalytic sites of trypsin by heparin was further confirmed by the significant reduction in the difference a bsorption spectra of proflavine. Both proteolytic and esterolytic acti vities of trypsin were shown to be markedly decreased by heparin, espe cially after 5 h incubation at 37 degrees C. However, when the proteol ytic and esterolytic activities of trypsin were measured on fresh solu tions not submitted to desalting treatments, variable activation inste ad of inhibition of both activities was observed in the presence of he parin, this effect waning spontaneously in time or after desalting tre atment. The paradoxical increase in functional activities was not inhi bited by soybean trypsin inhibitor and was accompanied by denaturation and fragmentation of the proteinase as demonstrated by spectroscopic analyses and SDS-PAGE of fresh solutions. The results obtained indicat e that heparin causes a rapid, time- and temperature-dependent conform ational alteration of trypsin with irreversible denaturation and degra dation of the proteinase. The underlying mechanism appears to be hepar in-catalyzed oxidative degradation of trypsin due to liberation of oxy gen radicals which are also responsible for the temporary increase in catalytic functions.