Study On The Effect Of The Small Molecules On The Part Properties Of The Trypsin

As an important digestive enzyme, the trypsin (E. C. 3. 4. 21. 4) has been widely used in food, pharmacy, leather, weave, cosmetic, farming, medical treatment and biology experiment. Therefore, in this paper the effect of the small molecules on the part properties of the trypsin was studied. The rutin was used to act on the trypsin, the effect of rutin on the trypsin was studied by UV differential spectrum and fluorescent spectrum. The effect of alditol, monosaccharide, oligosaccharide in different concentration, temperature, time and PH values on the enzyme's activity was studied. The effect of protective agent on the fluorescent spectrum of enzyme was also studied.The main conclusions of testing and experimental researches are concluded:1. In physiological conditions, UV absorption spectrum possesses weak positive absorption peak at 235 nm and strong absorption peak at 263 nm. The peak values gradually increase with the increasing concentration of rutin, while the wavelength of absorption peak keeps constant, this phenomena shows that rutin may change the conformation of trypsin, and leads the chromophore buried in the molecule exposed to the elements surface.2. When the wavelength of excitated light wave is 278 nm and the ratio of rutin and trypsin is 5/1, the biggest emission wavelength (λ_em=335.2nm) of the trypsin keep constant, but the relative fluorescence intensity decreases from 500.7 to 158.9. When the wavelength of excitated light wave is 295 nm, the red shift (0.2 nm) of theλ_em of the trypsin happened, theλ_em changed from 329.8nm to 330.0nm, however, the relative fluorescence intensity decreases from 274.6 to 114.7. These results indicate that the position of emission peaks doesn't change with the doping rutin, but the endogenesis fluorescence intensity regularly decrease with the increase of the concentration, that is, the rutin has an quenching effect on the fluorescence of trypsin.3. The Stern-Volmer quenching curve of rutin and trypsin was protracted by the fluorescence spectrum when the wavelength of excitated light wave is 295 nm. The linear equations (F₀/F=5.50×10⁴[Q]+1.01; K_q=5.50×10¹² (mol/L)^-1s^-1) were obtained by the Stern-Volmer quenching curve. It is assumed that the static state quenching is dominant. The binding constant K_A of rutin and trypsin was found to be 6.88×10⁴ (mol/L)^-1 and the number of binding site n was 1.02 by Lineweaver-Burk linear equation. So it is thought there is an binding site between the trypsin and rutin.4. The effect of alditol, monosaccharide, oligosaccharide in different concentration, temperature, time and PH values on the enzyme's activity was studied. The chosed groups with good stability and optimum concentration were 10% glycerin, 20% glycol, 3% glucose, 2% maltose, and 3% sucrose. At high temperature, the glycerin and glycol have safeguard function on the enzyme, but the function is still weak, however, the safeguard of glucose, maltose and sucrose on enzyme is relatively strong. The material was treated by protective agent of glucose at different PH conditions, PH stability is obviously better than those untreated, the stability range was broadened from 6 to 9. Therefore the simulated technical condition is realized, and the protective reagents which improve the stability of trypsin were compared and optimized, these experimental results provide theoretic data for the production and application of trypsin preparation.5. The effect of protective agent on the fluorescent spectrum of enzyme was studied. It is found that the red shift ofλ_em of trypsin which was disposed by protective agent mostly happens when the excitated light wave was applied, the relative fluorescence intensity decreases, but the effect is weak. These results show that the micro-circumstance polarity of tryptophan increases with doped protective agents, and the conformation of enzyme slightly changes.