MODULATION OF STABILITY PROPERTIES OF BOVINE TRYPSIN AFTER IN-VITRO STRUCTURAL-CHANGES WITH A VARIETY OF CHEMICAL MODIFIERS

Citation
R. Venkatesh et Pv. Sundaram, MODULATION OF STABILITY PROPERTIES OF BOVINE TRYPSIN AFTER IN-VITRO STRUCTURAL-CHANGES WITH A VARIETY OF CHEMICAL MODIFIERS, Protein engineering (Print), 11(8), 1998, pp. 691-698
Citations number
29
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biothechnology & Applied Migrobiology",Biology
Journal title
ISSN journal
0269-2139
Volume
11
Issue
8
Year of publication
1998
Pages
691 - 698
Database
ISI
SICI code
0269-2139(1998)11:8<691:MOSPOB>2.0.ZU;2-A
Abstract
Controlled chemical modification of enzymes, targeting groups not invo lved in the active site, can lead to modified catalysts that are intri nsically more efficient and resistant to heat and denaturing agents. B ovine pancreatic trypsin was covalently modified up to 75-85% with mon omeric glutaraldehyde (MGA), polymeric glutaraldehyde (PGA), oxidized sucrose and oxidized sucrose polymers (OSP 70 and OSP 400), Virtually no loss in activity occurred upon modification. Temperature optima of trypsin shifts from 45-76 degrees C and T-50 from 54-76 degrees C for the best modified sample made with OSP, The efficiency of the modifier s in stabilization was ranked in the order: OSP 400-T > OSP 70-T > PGA -T > MGA-T > Sucrose-T. Half-life of modified enzymes also followed th e same trend. Both stabilization factor and tilt decreased with increa sing temperatures. The free energy of activation for inactivation Delt a(Delta G not equal) varies from 12-20 kJ/mol and the activation entha lpy Delta(Delta H not equal) of the modified trypsin by 80-120 kJ/mol indicating stabilization. Inactivation of modified trypsin by urea is less noticeable. The character of the two-step inactivation process of trypsin changes with the degree of stabilization in that the duration of phase I one increased noticeably as stabilization increases. Nativ e trypsin fluoresces less intensely showing a red shift under the infl uence of denaturation, Such a fluorescence change is not so obvious fo r the modified enzymes indicating conformational stability acquired by modification.