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.

[The effect of ionizing radiation on enzymes. VI. The effect of gamma irradiation on the proteolytic activity of pepsin derived from porcine gastric mucosa]. / Ucinek ionizujícího zárení na enzymy. VI. Vliv zárení gama na proteolytickou úcinnost pepsinu z veprové zaludecní sliznice.

The present paper examines the effect of gamma radiation on the proteolytic effectiveness of pepsin of an activity of 6640, 8940 and 11,333 units in 1 g, expressed according to the Czechoslovak Pharmacopoeia. The samples were irradiated with graded doses from 5.2 to 128.7 kGy so that the course of inactivation could be determined. Effectiveness was determined six times in each sample and parallel experiments were statistically evaluated. The results are shown in Tables 1 to 3. The relative width of the interval of reliability only exceptionally exceeds 5%. A decrease in effectiveness shown in Fig. 1 shows that the course of the decrease in effectiveness did not depend in the interval used on the initial activity of the enzyme. In the semilogarithmic arrangement in Fig. 2, the dependence of the decrease in effectiveness on the dose possesses a linear course. The decrease in effectiveness in dependence on the dose of radiation can be approximately read in the graph in Fig. 3. The Czechoslovak Pharmacopoeia prescribes a dose of 24 kGy to achieve sterility by means of ionizing radiation. When microbiological indefectibility is the case, a smaller dose suffices, depending on the level of the initial contamination and the resistance of the present microorganisms. It usually does not exceed 10 kGy, in our case even 5 kGy. Nevertheless, there occurs a loss of effectivity, in the extreme case 10-13%. The largest dose used in the present paper decreased effectiveness by 60%. If ionizing radiation is to be used to decontaminate pepsin, a certain decrease in activity must be taken into account. In addition, a toxicological evaluation must be recommended.

Free radical reactions with proteins and enzymes: the inactivation of pepsin.

The reactions of free radicals produced by ionizing radiation with pepsin have been studied by steady-state inactivation measurements and by pulse radiolysis. In de-aerated solutions thehydroxyl radical has been found to be the most efficient of the primary free radicals generated from water in causing inactivation. The reactions of the more selective oxidizing inorganic radical anions Br2-. and (SCN)2-., with pepsin have also beenexamined. In the case of the thiocyanate radical anion (SCN)2-., the inactivation efficiency is found to depend on SCN- concentration, an effect shown to arise from a reversible redox reaction involving the tryptophan and (SCN)2-. radicals. The results demonstrate that tryptophan residue plays an essential role in the enzyme activity of pepsin.

[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.