Improving enzyme immobilization in mesocellular siliceous foams by microwave irradiation.

Microwave irradiation was used to immobilize papain and penicillin acylase in mesocellular siliceous foams (MCFs) at low temperature. The maximum loading of papain reached 984.1 mg/g, 1.26 times that obtained using the conventional, non-microwave-assisted method. The half-life (t(0.5)) of papain immobilized in MCFs by microwave irradiation at 80 degrees C was 17 h, 5.21 times that of papain immobilized by conventional means. The activities of papain and penicillin acylase immobilized with the microwave-assisted method were 779.6 U/mg and 141.8 U/mg respectively, 1.86 and 1.39 times of those obtained without microwave immobilization. Using microwave irradiation it only took 140 s for penicillin acylase, an enzyme of large dimensions, to be immobilized in MCFs. In contrast, it took 15 h to do the same using the conventional method. The results showed that microwave irradiation improved the adsorption and immobilization of enzymes in mesocellular siliceous foams.

Light-induced inhibition of papain by a {Mn-NO}6 nitrosyl: identification of papain-SNO adduct by mass spectrometry.

Modification of Cys25 at the active site of the cysteine protease papain by S-nitrosylation inhibits its hydrolytic ability. Previous studies have demonstrated that NO donors N-nitrosoanilines inhibit papain activity via formation of S-NO bond formation at the active site while NO donors such as S-nitroso-N-acetyl-penicillamine (SNAP), N-nitrosoaniline derivatives, and S-nitroso-glutathione (GSNO) inhibit the enzyme via S-thiolation by thiyl radicals generated from the S-nitrosothiols. In this study, we report papain inactivation by a photosensitive {Mn-NO}(6) nitrosyl [(PaPy(3))Mn(NO)](ClO(4)) (1) where PaPy(3)(-) is the anion of the designed ligand N,N-bis(2-pyridylmethyl)amine-N-ethyl-2-pyridine-2-carboxamide. This nitrosyl releases NO upon exposure to visible light of low intensity (50W tungsten lamp). With N(alpha)-benzoyl-l-arginine-p-nitroanilide (l-BApNA) as the substrate, the dissociation constant for the breakdown of the enzyme-inactivator complex (K(I)) and the overall inactivation rate constant (k(i)) were calculated to be 2.46mM and 64.8min(-1), respectively. The papainS-NO adduct has been identified using electrospray mass spectrometry (ESI-MS). The results demonstrate that controlled inactivation of papain can be achieved with the {Mn-NO}(6) nitrosyl 1 and light. The reaction is clean and the extent of inactivation is directly proportional to the exposure time.

[Modulation of the intensity of fluorescence of aqueous solutions of proteins by magnetic field]. / Moduliatsiia intensivnosti fluorestsentsii vodnykh rastvorov belkov magnitnym polem.

Effect of the constant magnetic field with up to 3.2 X 10(-4) A/m intensity on the fluorescence of papain aqueous solutions was investigated. It has been shown that depending on the magnetic field direction a reversible decrease or increase of fluorescence intensity takes place. The variation of fluorescence intensity under the influence of magnetic field is maximal under excitation at long wave ultra-violet light. The effect increases with the increase of temperature, increases linearly with the increase of magnetic field intensity but doesn't depend on protein concentration in diluted solutions. The examination of the data leads to the conclusion on the existence of two possible mechanisms: the variation of properties of surface tryptophan residues environment and paramagnetic orientation of protein globule under the influence of a magnetic field.

The inactivation of papain by high LET radiations.

The effect of varying LET over a wide range (0.2-1570 eV/nm) on the radiation-induced inactivation of the enzyme papain in dilute aqueous solution has been investigated. Measurements of total, reparable and non-reparable inactivation G values in oxygen, nitrous oxide and argon saturated solutions have allowed the contributions to inactivation from radicals and hydrogen peroxide to be evaluated. At high LET the results demonstrate an increasing component due to reaction of the superoxide radical, formed from oxygen produced in the track as a primary radiolysis product. This effect was not observed in our previous study with ribonuclease due to the insensitivity of ribonuclease to inactivation by superoxide and hydrogen peroxide. The results obtained with papain clearly demonstrate a maximum in G (H2O2) at an LET of approximately 140 eV/nm. Generation of O2 within the track as a primary radiolysis product at high LET now appears to be confirmed as an important mechanism leading to reduction in the oxygen enhancement ratio for cellular systems exposed to high LET radiations (Baverstock and Burns 1981).

Behavior of enzyme activity in immobilized proteases.

Proteases such as trypsin, alpha-chymotrypsin, papain, and thermolysin were immobilized by radiation polymerization of various monomers at low temperatures, and behavior of enzyme activity in immobilized proteases was studied. The enzyme activity in immobilized proteases appeared to be different by the kind of proteases; the order of the magnitude of the enzyme activity was papain greater than trypsin greater than thermolysin greater than alpha-chymotrypsin. This difference of the enzyme activity was explained by the change of the molecular conformation in enzyme reaction.

Radiolytic and enzymatic dimerization of tyrosyl residues in insulin, ribonuclease, papain and collagen.

Insulin, ribonuclease, papain and collagen solutions saturated with nitrogen, N2O or air were irradiated with doses of 10 to 640 Gy of gamma rays. Protein solutions were also oxidized enzymatically in a system of horse-radish peroxidase: hydrogen peroxide. Column chromatography (Sephadex G-75 or Sephacryl S-200) of treated protein solutions revealed that they contain protein molecular aggregates. Nitrogen saturation of solution before irradiation was most favourable for radiation-induced aggregation of proteins. Fluorescence analysis of protein solutions resulted in detection of dityrosyl structures in irradiated as well as in enzymatically oxidized proteins. Concentration of dityrosine in proteins studied was determined fluorimetrically in their hydrolysates separated on BioGel P-2 column. In irradiated proteins, dityrosine was present almost exclusively in their aggregated forms. In proteins oxidized enzymatically, dityrosine was also present in fractions containing apparently unchanged protein. Mechanisms which could account for differences in the yield of dityrosine formation in radiolysis and in enzymatic oxidation of proteins are suggested.

Effects of superoxide dismutase, dithiothreitol and formate ion on the inactivation of papain by hydroxyl and superoxide radicals in aerated solutions.

Losses in enzyme activity and sulphydryl content have been studied in aerated papain solutions containing formate, superoxide dismutase and dithiothreitol. Both formate and dithiothreitol converted .OH to .O2-, whereas superoxide dismutase completely suppressed the inactivation by .O2-. Using results from all three systems, the fraction of .O2- reactions with papain that caused inactivation of the enzyme was 0.33 +/- 0.07. The results also showed that the fraction of .OH reactions, which cause inactivation of papain, is significantly higher in aerated than in oxygen-free solutions.

Conversion of the active-site cysteine residue of papain into a dehydro-serine, a serine and a glycine residue.

Photolysis of papain which had been inhibited with 2-bromo-2',4'-dimethoxyacetophenone regenerated papain, but also formed [deltaSer25]-papain (i.e. papain in which the active-site cysteine residue 25 was replaced by dehydroserine) via the intermediate dehydrocysteine analogue, [deltaCys25]-papain. Reduction with sodium borohydride gave [Ser25]papain. Both [Ser25]papain and [deltaSer25]-papain had binding properties similar to those of papain, but were devoid of enzymic activity. Their fluorescence properties were also investigated. Incubation of [deltaSer25]papain at pH 9.0 gave [Gly25]papain.

[Luminescence quenching of UV-irradiated proteins]. / Tushenie liuminestsentsii belkov pri UF-obluchenii

Kinetic curves of paramagnetic centres accumulation and quenching of the luminescence of UV-irradiated proteins at 77 degrees K are compared. Initiation of luminescence of UV-irradiated proteins at the annealing of paramagnetic centres was studied. The luminescence of UV-irradiated proteins was shown to be quenched with the paramagnetic centres formed. It was shown that protein absorption spectrum was not changed when the limiting concentration of paramagnetic centres was obtained in it.

Photochemical inactivation of enzymes.

The mechanisms of enzyme inactivation by ultraviolet light and visible light in the presence of sensitizing dyes are reviewed. Recent flash photolysis studies on amino acids and enzymes are summarized in terms of proposed models relating the initial photochemical reactions to permanent chemical and biological damage. The generation and reactions of singlet oxygen are discussed in connection with photodynamic processes. The photochemical results are compared with ionizing radiations, particularly pulse radiolytic methods employing radical anions as selective probes. The interrelationships between the various modes of enzyme inactivation are discussed, as well as the new information to be learned about the structure and functions of the native enzymes from selective radiation-induced alterations.

[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.