Biophysical evaluation of milk-clotting enzymes processed by high pressure.

High pressure processing (HPP) is able to promote changes in enzymes structure. This study evaluated the effect of HP on the structural changes in milk-clotting enzymes processed under activation conditions for recombinant camel chymosin (212MPa/5min/10°C), calf rennet (280MPa/20min/25°C), bovine rennet (222MPa/5min/23°C), and porcine pepsin (50MPa/5min/20°C) and under inactivation conditions for all enzymes (600MPa/10min/25°C) including the protease from Rhizomucor miehei. In general, it was found that the HPP at activation conditions was able to increase the intrinsic fluorescence of samples with high pepsin concentration (porcine pepsin and bovine rennet), increase significantly the surface hydrophobicity and induce changes in secondary structure of all enzymes. Under inactivation conditions, increases in surface hydrophobicity and a reduction of intrinsic fluorescence were observed, suggesting a higher exposure of hydrophobic sites followed by water quenching of Trp residues. Moreover, changes in secondary structure were observed (with minor changes seen in Rhizomucor miehei protease). In conclusion, HPP was able to unfold milk-clotting enzymes even under activation conditions, and the porcine pepsin and bovine rennet were more sensitive to HPP.

[Influence of heavy water (D20) on the conformation and UV-sensitivity of proteins]. / Vliianie tiazheloi vody (D20) na konformatsiiu i UF-chvstvitel'nost' belkov.

The heavy water (D2O) has been shown to induce the conformational transitions in trypsin, chymotrypsin and pepsin. The transfer of proteins from H2O into D2O results a change in their sensitivity to UV-light. An increase in sensitivity to the irradiation at 248 nm and a decrease in sensitivity to the irradiation at 280 nm were observed. The quantum yield of chromophore photolysis (for cystyne and tryptophan) is correspondingly changed. However, although the quantum yield of sensitized reduction of cystine by solvated electrons photochemically ejected from the aromatic acid residues during irradiation at 280 nm increases instead of a rise a drop in the quantum yield of protein inactivation is registered. The data obtained are discussed in terms of importance of solvated shell for conformational stability of proteins. The solvated electrons are suggested to be transfered mainly to nonessential disulfide bridges within trypsin molecule. Rupture of these bonds does not result in trypsin inactivation.

[Kinetic equation for the process of accumulation of paramagnetic centers in proteins exposed to UV-radiation]. / Kineticheskoe uravnenie protsessa nakopleniia paramagnitnykh tsentrov v proteinakh pri deistvii UF-sveta

The process of accumulation of paramagnetic centres in UV-irradiated solutions of simple proteins at 77 degrees K has been studied. A kinetic equation describing the accumulation of radicals in protein is obtained. Experimentally obtained curves of radical accumulation coincide with the theoretical ones.

[Mechanism of the formation of paramagnetic centers in proteins exposed to UV-radiation]. / Mekhanizm obrazovaniia paramagnitnykh tsentrov v belkakh pri deistvii UF-sveta

It is shown that the process of formation of paramagnetic centres in UV-irradiated at 77degreesK protein solutions in a biquantum one. The protein molecule in triplet state is an intermediate product.

[Accumulation of paramagnetic centers in protein solutions exposed to UV-radiation]. / Issledovanie nakopleniia paramagnitnykh tsentrov v rastvorakh belkov pri deistvii UF-sveta

It is shown that concentration of paramagnetic centres (PC) in UV-irradiated protein solutions at 77degreesK approximates the limiting value. The limiting number of PC (n) per one molecule is in direct proportion to that of aromatic amino acid residues in it n(sigma)=2+0,1 sigma. The formation of PC slopps because all the energy absorbed by aromatic amino acid residues is transfered to the radicals formed.