Modulation of stability properties of bovine trypsin after in vitro structural changes with a variety of chemical modifiers.
Controlled chemical modification of enzymes, targeting groups not involved in the active site, can lead to modified catalysts that are intrinsically more efficient and resistant to heat and denaturing agents. Bovine pancreatic trypsin was covalently modified up to 75-85% with monomeric 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 T50 from 54-76 degrees C for the best modified sample made with OSP. The efficiency of the modifiers 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 the same trend. Both stabilization factor and t1/2 decreased with increasing temperatures. The free energy of activation for inactivation delta(deltaG*) varies from 12-20 kJ/mol and the activation enthalpy delta(deltaH*) 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. Native trypsin fluoresces less intensely showing a red shift under the influence of denaturation. Such a fluorescence change is not so obvious for the modified enzymes indicating conformational stability acquired by modification.
Venkatesh
R
R
Centre for Protein Engineering and Biomedical Research, The Voluntary Health Services, Madras, India.
Sundaram
P V
PV
D000818:Animals; D002417:Cattle; D004795:Enzyme Stability; D005976:Glutaral / Q000031:analogs & derivatives / Q000737:chemistry; D006207:Half-Life; D006358:Hot Temperature; D007700:Kinetics; D008623:Mercaptoethanol / Q000737:chemistry / Q000378:metabolism; D011108:Polymers / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism; D014357:Trypsin / Q000737:chemistry* / Q000378:metabolism*; D014508:Urea / Q000737:chemistry / Q000378:metabolism
D011108:Polymers; C022841:poly(glutaraldehyde); D013395:Sucrose; D008623:Mercaptoethanol; D014508:Urea; D014357:Trypsin; D005976:Glutaral