Surface Of Protein Globule Research Articles

Sensitizing effect of Z,Z-bilirubin IXα and its photoproducts on enzymes in model solutions

In model systems, we have studied side effects which may be induced by light during phototherapy of hyperbilirubinemia (jaundice) in newborn infants, with the aim of reducing the Z,Z-bilirubin IXα (Z,Z-BR IXα) level. We have shown that the sensitizing effect of Z,Z-BR IXα, localized at strong binding sites of the human serum albumin (HSA) macromolecule, is primarily directed at the amino acid residues of the carrier protein and does not involve the molecules of the enzyme (lactate dehydrogenase (LDH)) present in the buffer solution. The detected photodynamic damage to LDH is due to sensitization by bilirubin photoisomers, characterized by lower HSA association constants and located (in contrast to native Z,Z-BR IXα) on the surface of the HSA protein globule. Based on study of the spectral characteristics of the photoproducts of Z,Z-BR IXα and comparison of their accumulation kinetics in solution and the enzyme photo-inactivation kinetics, we concluded that the determining role in sensitized damage to LDH is played by lumirubin. The photosensitization effect depends on the wavelength of the radiation used for photoconversion of bilirubin. When (at the beginning of exposure) we make sure that identical numbers of photons are absorbed by the pigment in the different spectral ranges, the side effect is minimal for radiation corresponding to the long-wavelength edge of the bilirubin absorption band. We have shown that for a bilirubin/HSA concentration ratio >2 (when some of the pigment molecules are sorbed on the surface of the protein globule), the bilirubin can act as a photosensitizing agent for the enzyme present in solution. We discuss methods for reducing unfavorable side effects of light on the body of newborn infants during phototherapy of hyperbilirubinemia.

Surface hydrophobic amino acid residues in cellulase molecules as a structural factor responsible for their high denim-washing performance

The denim-washing performance of six purified fungal cellulases (four endo-1,4-beta-D-glucanases and two cellobiohydrolases) was compared using a model microassay. The performance of cellobiohydrolases per mg of protein was much lower than that of endoglucanases. For endoglucanases, it varied up to 5 times between the best and the worst enzyme. Experiments with amino acids immobilized on cross-linked agarose showed that their side chains may bind indigo owing to hydrophobic interactions and formation of hydrogen bonds. The best binding effects provided Tyr and Phe. Analysis of three-dimensional structures of cellulase molecules showed that a certain correlation exists between the washing performance of enzyme and (i) quantity (percentage) of aromatic residues exposed to solvent on the surface of protein globule or (ii) overall percentage of the surface hydrophobic residues. Data presented provide an evidence that the molecules of certain cellulases, which have hydrophobic domains (clusters of closely located non-polar residues) on their surface, may bind indigo and thus act as emulsifiers helping the dye to float out of cellulose fibers to the bulk solution.

The Electron-Transfer Reactions of NADPH-Cytochrome P450 Reductase with Nonphysiological Oxidants

The steady-state kinetics of oxidation of rat liver NADPH:cytochrome P450 reductase (EC 1.6.2.4) by quinones, aromatic nitrocompounds, ferricyanide, Fe(EDTA) −, and cytochrome c has been studied. The logarithms fo bimolecular rate constants of reduction ( k cat/ K m ) of quinones and nitrocompounds increase with the increase in their single-electronreduction potential ( E 1 7), reaching a maximum value at E 1 7 > −0.15 V. The reactivities of nitroaromatics are about by an order of magnitude lower than the reactivities of quinones. For a series of nitroaromatics including the compounds with previously undetermined E 1 7 values, an orthogonality was found between their reactivities toward cytochrome P450 reductase, flavocytochrome b 2 (EC 1.1.2.3), and the NADPH:adrenodoxin reductase (EC 1.18.1.2)-adrenodoxin system. This indicates the absence of significant specific interactions during these reactions. The effects of ionic strength on reaction kinetics and the character of inhibition by a product of reaction, NADP +, are in accordance with the reduction of oxidants at the negatively charged site in the surroundings of FMN of P450 reductase. Quinones inactivate oxidized reductase modifying the NADP(H) binding site. The redox cycling of quinones markedly slows the inactivation. The kinetic data presented are consistent with an outer-sphere electron transfer mechanism. The analysis of kinetics of reduction of cytochrome c, ferricyanide, and Fe(EDTA) − using the model of Mauk et al. (A. G. Mauk, R. A. Scott, and H. B. Gray (1980) J. Am. Chem. Soc. 102, 4360-4363) gives calculated distances of FMN from the surface of protein globule, 0.33-0.63 nm. The data from nitroreductase reactions of cytochrome P450 reductase, flavocytochrome b 2, and adrenodoxin were used for approximate evaluation of previously unknown E 1 7 of nitrocompounds.

Possible conformation of interferons: A prediction based on amino acid composition and sequence

Using the method of estimation of α-helical and β-folded types of secondary structure from amino acid composition, it has been established that interferons contain about 60–70% of α-helices and not more than 10–20% of β-structure. The same result was obtained using empirical and stereochemical rules for the prediction of protein secondary structure of amino acid sequence. The hydrophobic clusters on the surfaces of the α-helical segments were determined. Using the method for packing of α-helical segments into the globule suggested by Efimov, and taking into account the disulfide bond arrangement in leucocyte interferon, two alternative globular conformations with prevailing α-helix were obtained. The analysis of these conformations shows an anomalous distribution of charged amino acid residues conserved in the primary structure of leucocyte and fibroblast interferons. Nine positively charged amino acid residues are concentrated in one site on the surface of protein globule. It is possible that this site is responsible for some general biological activity of the interferons.