Biphasic pattern of heparin-induced oxidative degradation of trypsin in the presence of glucose.
The present work tested the hypothesis that the oxidative effects of heparin on trypsin may be modified by the addition of another reducing substance. Glucose was chosen as reducing sugar and used at a concentration (15 mM) and an incubation time (5 h at 37 degrees C) with trypsin which may cause only modest oxidative modifications, thus resembling what happens in vivo in the presence of slight, long-lasting hyperglycaemia. The effects of 10, 100 and 200 micrograms/mL standard heparin on both structural and functional properties of glucose-treated bovine trypsin were measured by UV and fluorescence emission spectra and by the proteolytic and esterolytic activities of trypsin on two different substrates. Radical formation was measured by the reduction of nitroblue tetrazolium and acetylated cytochrome c. The influence of incubation and desalting of solutions were also evaluated. In the absence of heparin but in the presence of glucose, no gross structural alteration of trypsin was observed, nor was any fragmentation of the enzyme visible in the electrophoretic pattern. However, despite the fact that soluble free radicals were not detectable, glucose did induce the formation of reactive species linked to trypsin itself, as confirmed by the findings of increased reduction of nitroblue tetrazolium by fresh undesalted, and incubated desalted glucose-treated trypsin solutions. Heparin, mostly at 10 micrograms/mL (which caused an increase of 9.64 nmol reduced cytochrome c/mg protein per min) significantly changed both the nature and concentration of the radicals which were formed. Heparin-induced oxygen radical production was rapid in onset, and markedly modified the structure and function of the enzyme which in time underwent complete inactivation and fragmentation. At both 100 and 200 micrograms/mL, heparin appeared to slow the oxidative process, as spectroscopic analysis and electrophoretic pattern showed less profound structural modifications of trypsin. Overall results show that heparin-induced oxidation of trypsin follows a biphasic, concentration-dependent pattern in the presence of another reducing substance such as glucose. Low heparin concentrations enhance the oxidative potential of glucose, whereas higher concentrations antagonize it.
Finotti
P
P
Department of Pharmacology, University of Padova, Italy.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002384:Catalysis / Q000187:drug effects; D002417:Cattle; D003574:Cytochrome c Group / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D006493:Heparin / Q000494:pharmacology*; D009580:Nitroblue Tetrazolium / Q000378:metabolism; D010084:Oxidation-Reduction / Q000187:drug effects; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013552:Swine; D014357:Trypsin / Q000187:drug effects* / Q000378:metabolism*; D014361:Trypsin Inhibitors / Q000494:pharmacology
D000596:Amino Acids; D003574:Cytochrome c Group; D014361:Trypsin Inhibitors; D009580:Nitroblue Tetrazolium; D006493:Heparin; D014357:Trypsin; D005947:Glucose