Differential mechanisms for structural and functional alterations of trypsin by heparin, evidence for a specific, radical-generating mechanism at low heparin concentrations.
The oxidative mechanism whereby heparin may interact with various proteins was investigated in detail in this work by addressing the role of doses of heparin on the nature and effects of its binding to bovine trypsin, taken as reference protein. Unfractionated heparin was used at concentrations ranging from 6 to 400 microg/ml with a fixed trypsin concentration (250 microg/ml). At concentrations of up to 60 microg/ml, equivalent to trypsin/heparin molar ratios of between 30 and 3, increasing inhibition of amidolytic activity and radical-dependent peptide bond cleavage of the enzyme was observed, with the appearance in the electrophoretic pattern of new bands of trypsin fragments to which heparin was demonstrated to be bound specifically. Structural modifications were also revealed by increases in fluorescence emission spectra. On the whole, however, the alterations induced by these heparin concentrations only involved a limited number of trypsin molecules. At concentrations from 120 to 400 microg/ml (equivalent trypsin/heparin molar ratios of 1.5-0.46), heparin binding to trypsin appeared to cause more profound and generalized alterations of enzyme structure and function, with dose-dependent quenching of fluorescence emission and almost complete loss of amidolytic activity, although evidence of radical production was lacking. Collectively, the results stress the crucial role of heparin dose on both the nature and effects of its binding to trypsin. The change in heparin effects which reflects distinct underlying molecular mechanisms occurs dramatically at a critical concentration threshold. While a specific, radical-generating mechanism operates at low concentrations, less specific ionic linkages, apparently independent of radical production, best explain the effects of high heparin concentrations.
Finotti
P
P
Department of Pharmacology, University of Padova, Italy. finpa@ux1.unipd.it
Corvaja
C
C
Pagetta
A
A
D000818:Animals; D002417:Cattle; D004305:Dose-Response Relationship, Drug; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals; D006493:Heparin / Q000008:administration & dosage / Q000378:metabolism / Q000494:pharmacology*; D019763:Heparin Lyase / Q000494:pharmacology; D010446:Peptide Fragments / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*
D005609:Free Radicals; D010446:Peptide Fragments; D006493:Heparin; D014357:Trypsin; D019763:Heparin Lyase