Structural and functional modifications of bovine trypsin by heparins. Influence of heparin molecular mass and structure.
Heparins with different structures, charge density and molecular mass were evaluated for their capacity to induce structural and functional alterations of bovine trypsin in a low ionic strength buffer (20 mM Tris-HCl pH 7.4). Unfractionated heparin, and slow and fast moving heparin species increased the fluorescence peak emission of trypsin to the same extent (about +40.0%), whilst partially desulfated and re-N-acetylated heparin with a charge density of 1.47 modified the fluorescence at 330 nm by about +27% and natural heparan sulfate with a sulfate-to-carboxyl ratio < 1 by about +13%. Heparin fractions with narrow polydispersity and the same charge density (produced by chemical depolymerization in the presence of free radicals and further gel-permeation chromatography) having molecular mass lower than about 6000 interact with trypsin to a less extent, even though fractions with molecular mass of about 4500 and 3600 partially retain this property. No modification of fluorescence peak emission of trypsin with heparin was appreciable when the ionic strength of the buffer was increased to 0.3 mM NaCl. An altered ability to reduce cytochrome c was observed for heparins of different charge density; fragments with molecular mass lower than approximately 4000 were also unable to produce superoxide. Trypsin was degraded into fragments by heparin and derivatives after 3 h incubation at 37 degrees C. After electrophoresis in polyacrylamide-gels the trypsin bands disappeared and fragments with lower molecular mass were more evident. This effect depended on the molecular mass of heparin, and was more evident for unfractionated heparin and for a heparin fraction with a molecular mass of 7820. The esterolytic activity of trypsin was inhibited to the same extent by heparin derivatives of various structure and charge density while activity undermet minor changes in the presence of heparin fractions of Mr lower than 4000.
Volpi
N
N
Department of Biologia Animale, University of Modena, Italy. volpi@unimo.it
D000818:Animals; D002417:Cattle; D004950:Esterases / Q000378:metabolism; D006493:Heparin / Q000737:chemistry / Q000494:pharmacology*; D006497:Heparan Sulfate / Q000494:pharmacology; D007700:Kinetics; D013050:Spectrometry, Fluorescence; D013481:Superoxides / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism
D004950:Esterases; D006493:Heparin; D006497:Heparan Sulfate; D013481:Superoxides; D014357:Trypsin