IR spectroscopy in the research of UV modification processes for immobilized trypsin

Abstract

Today the solution of theoretical and practical issues related to understanding the mechanism of UV light action on biosystems of various organization levels, patterns of UV induced changes in the structural and functional characteristics of immobilized enzymes remains relevant. In this regard, the study of the physical and chemical properties of proteases under the influence of UV light must be continued and developed. We have investigated the mechanism of the UV light action on trypsin immobilized on the chitosan matrix, since for immobilized enzymes, the number of possible inactivating pathways is substantially less than in the case of soluble proteins.
Two types of chitosan served as carriers for immobilization: medium molecular weight (200 kDa) and high molecular weight (350 kDa). This polymer has biocompatibility, low toxicity and nonimmunogenicity, which causes widespread use of chitosan in medicine and pharmacology. Immobilization of
trypsin samples were adsorption method. Determination of the protein amount and activity of trypsin was performed using a modified Lowry method. Statistical processing of the results was performed using student's t-test at p<0.05.
It was found that the enzymatic activity of immobilized trypsin under the influence of UV light substantially is not subject to change. It is suggested a photoprotective effect of chitosan matrix. For a more detailed study of photoprocesses originating from the adsorbed chitosan matrix with trypsin under the action of UV light were investigated IR-spectra of the immobilized enzyme after exposure to UV-irradiation at dosesof 151-6040 J/m2. It was revealed that at a dose of 6040 J/m² there are changes in the structure of the protein complex, associated with varying the number of disordered structures and β-layers in the enzyme molecule, –COO– amino acid fluctuations, manifested in a decrease in the activity of the drug. The study of the mechanism of ultraviolet light action on biological systems of different organization levels, including the enzyme, today is one of the central problems of biophysics and physical chemistry biology and reveals mechanisms destructive-modifying, regulatory and therapeutic and prophylactic effect UV-radiation