title: Role of reducing terminals in unfractionated and low-molecular-mass heparins in causing free radical generation and loss of structure and activity of trypsin.
abstract: The role of both the length of saccharide chain and reducing terminals in the heparin molecule in causing oxidative effects on proteins was investigated by employing unfractionated and low-molecular-mass heparins (LMMH), with both intact and reduced reducing terminals on bovine trypsin. The effects of heparin were found to be dependent on both the concentration and time of incubation. Heparins with intact reducing terminals caused significantly higher structural and functional alterations of trypsin compared with heparins with reduced reducing terminals. LMMH was slightly more effective than unfractionated heparin (UNFH) in reducing structural integrity and inhibiting the amidolytic activity of trypsin when used at the same mass, but not molar concentrations. Neither the length of saccharide chains nor the number of intact reducing terminals on the heparin molecule appeared to influence the characteristics of the initial binding of heparin to trypsin, but both these variables crucially affected linkages which in time mediate the inhibition of catalytic activity and the formation of free radicals, ultimately responsible for peptide bond cleavage in trypsin. The results suggest that both a critical number of saccharide units, preferentially lying on shorter chains, and intact reducing terminals in the heparin molecule are involved in setting up the binding which generates radicals and leads to loss of structure and function of the proteinase.
authors.affiliation: Department of Pharmacology, University of Padova, Italy. finpa@ux1.unipd.it
authors.forename: P
authors.initials: P
authors.lastname: Finotti
authors.forename: A
authors.initials: A
authors.lastname: Pagetta
authors.forename: C
authors.initials: C
authors.lastname: Corvaja
mesh_terms: D000818:Animals; D002417:Cattle; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D005609:Free Radicals; D006493:Heparin / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006495:Heparin, Low-Molecular-Weight / Q000737:chemistry* / Q000378:metabolism* / Q000494:pharmacology; D008958:Models, Molecular; D010446:Peptide Fragments / Q000032:analysis / Q000737:chemistry; D011487:Protein Conformation; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry* / Q000187:drug effects / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D006495:Heparin, Low-Molecular-Weight; D010446:Peptide Fragments; D006493:Heparin; D014357:Trypsin