Functional properties of partially oxidized trout hemoglobins.

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.     

Main chain and side chain dynamics of oxidized flavodoxin from Cyanobacterium anabaena.

Oxidized flavodoxin from Cyanobacterium anabaena PCC 7119 is used as a model system to investigate the fast internal dynamics of a flavin-bearing protein. Virtually complete backbone and side chain resonance NMR assignments of an oxidized flavodoxin point mutant (C55A) have been determined. Backbone and side chain dynamics in flavodoxin (C55A) were investigated using (15)N amide and deuterium methyl NMR relaxation methods. The squared generalized order parameters (S(NH)(2)) for backbone amide N-H bonds are found to be uniformly high ( approximately 0.923 over 109 residues in regular secondary structure), indicating considerable restriction of motion in the backbone of the protein. In contrast, methyl-bearing side chains are considerably heterogeneous in their amplitude of motion, as indicated by obtained symmetry axis squared generalized order parameters (S(axis)(2)). However, in comparison to nonprosthetic group-bearing proteins studied with these NMR relaxation methods, the side chains of oxidized flavodoxin are unusually rigid.     

Kinetic studies on oxidized and partially reduced cytochrome c oxidase.

1. Kinetic studies have been performed with beef-heart cytochrome c oxidase, with the enzyme either in its oxidized, resting state or pretreated anaerobically with different amounts of reduced cytochrome c. The techniques used for the study have been stopped-flow spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. 2. The results show that the one-electron equivalent-reduced enzyme rapidly oxidizes one further equivalent of aerobically or anaerobically added ferrocytochrome c, with a rate constant of 5 . 10(6) M-1 . s-1. 3. When an excess of ferrocytochrome c in the presence of oxygen is added to the one-electron-reduced enzyme, the same turnover rate is obtained as in experiments with the resting enzyme. 4. The one-electron equivalent-enzyme reacts with CO with a rate constant of 4 . 10(4) M-1 . s-1 to yield approx. 35% of the CO compound as compared with the reaction between the fully reduced enzyme and CO. 5. It is shown that on reduction the enzyme is converted into an active form, but it is concluded that the enzyme does not have to be fully reduced before it is catalytically active.     

Degradation of partially oxidized alginate and its potential application for tissue engineering

Alginate has been widely used in a variety of biomedical applications including drug delivery and cell transplantation. However, alginate itself has a very slow degradation rate, and its gels degrade in an uncontrollable manner, releasing high molecular weight strands that may have difficulty being cleared from the body. We hypothesized that the periodate oxidation of alginate, which cleaves the carbon-carbon bond of the cis-diol group in the uronate residue and alters the chain conformation, would result in promoting the hydrolysis of alginate in aqueous solutions. Alginate, oxidized to a low extent (approximately 5%), degraded with a rate depending on the pH and temperature of the solution. This polymer was still capable of being ionically cross-linked with calcium ions to form gels, which degraded within 9 days in PBS solution. Finally, the use of these degradable alginate-derived hydrogels greatly improved cartilage-like tissue formation in vivo, as compared to alginate hydrogels.     

Oxygen binding to partially oxidized hemoglobin. Analysis in terms of an allosteric model

We report on oxygen binding to partially oxidized (aquomet) hemoglobin. The fractional saturation with oxygen is evaluated by deconvoluting the optical absorption spectra, in the 500-700 nm wavelength region, in terms of oxyhemoglobin, deoxyhemoglobin and methemoglobin spectral components. Experiments have been performed with auto-oxidized samples and with samples obtained by mixing ferrous hemoglobin with fully oxidized hemoglobin (mixed samples). An increase in oxygen affinity and a decrease in cooperativity are observed on increasing the amount of ferric hemoglobin in the sample. A high cooperativity (nH approximately 2) is maintained even in the presence of 50-60% ferric hemes. Moreover, for equal amounts of methemoglobin the oxygen affinity is lower and the cooperativity higher for mixed samples than for those auto-oxidized. The results are analyzed within the framework of a modified Monod-Wyman-Changeux allosteric model taking into account the effects brought about by the presence of oxidized hemes and of alpha betta dimers. The distribution of ferric subunits within the tetramers in fully deoxygenated and fully oxygenated samples, as derived from the model, provides details on the cooperative behavior of partially oxidized hemoglobin.     

The crystal structure of Cupriavidus necator nitrate reductase in oxidized and partially reduced states

The periplasmic nitrate reductase (NapAB) from Cupriavidus necator is a heterodimeric protein that belongs to the dimethyl sulfoxide reductase family of mononuclear Mo-containing enzymes and catalyzes the reduction of nitrate to nitrite. The protein comprises a large catalytic subunit (NapA, 91 kDa) containing the molybdenum active site plus one [4Fe-4S] cluster, as well as a small subunit (NapB, 17 kDa), which is a diheme c-type cytochrome involved in electron transfer. Crystals of the oxidized form of the enzyme diffracted beyond 1.5 Å at the European Synchrotron Radiation Facility. This is the highest resolution reported to date for a nitrate reductase, providing true atomic details of the protein active center, and this showed further evidence on the molybdenum coordination sphere, corroborating previous data on the related Desulfovibrio desulfuricans NapA. The molybdenum atom is bound to a total of six sulfur atoms, with no oxygen ligands or water molecules in the vicinity. In the present work, we were also able to prepare partially reduced crystals that revealed two alternate conformations of the Mo-coordinating cysteine. This crystal form was obtained by soaking dithionite into crystals grown in the presence of the ionic liquid [C₄mim]Cl⁻. In addition, UV-Vis and EPR spectroscopy studies showed that the periplasmic nitrate reductase from C. necator might work at unexpectedly high redox potentials when compared to all periplasmic nitrate reductases studied to date.     

Preparation and properties of partially oxidized N-methylisoquinolinium bis(oxalato)platinate nanowires

Electrochemical oxidation of [C₁₀H₁₀N]₂[Pt(ox)₂] · H₂O (1) (C₁₀H₁₀N = N-methylisoquinolinium, ox = oxalate) leads to the synthesis of new partially oxidized platinum nanowires of formula [C₁₀H₁₀N]_1.6[Pt(ox)₂] · 3H₂O (2). The nanowires were characterized by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). Wires with diameters of less than 5 nm and lengths of over 1 μm were observed. Energy dispersive spectroscopy (EDS) and microanalysis confirmed the degree of partial oxidation of the nanowires. The bulk electrical properties including phase angle and real and imaginary impedance values were measured and a model of the electrical conduction circuit was proposed. The significance of this work is that the large N-methylisoquilolinium cation leads to nanostructures, possibly involving individual molecular wires which has not been previously observed in the bis(oxalato)platinate nanowire system.     

Oxidatively modified fatty acyl chain determines physicochemical properties of aggregates of oxidized phospholipids

In vivo oxidation of glycerophospholipid generates a variety of products including truncated oxidized phospholipids (tOx-PLs). The fatty acyl chains at the sn-2 position of tOx-PLs are shorter in length than the parent non-oxidized phospholipids and contain a polar functional group(s) at the end. The effect of oxidatively modified sn-2 fatty acyl chain on the physicochemical properties of tOx-PLs aggregates has not been addressed in detail, although there are few reports that modified fatty acyl chain primarily determines the biological activities of tOx-PLs. In this study we have compared the properties of four closely related tOx-PLs which differ only in the type of modified fatty acyl chain present at the sn-2 position: 1-palmitoyl-2-azelaoyl-sn-glycero-3-phosphocholine (PazePC), 1-palmitoyl-2-(9′-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC), 1-palmitoyl-2-glutaroyl-sn-glycero-3-phosphocholine (PGPC), and 1-palmitoyl-2-(5′-oxo-valeroyl)-sn-glycero-3-phosphocholine (POVPC). Aggregates of individual tOx-PL in aqueous solution were characterized by fluorescence spectroscopy, size exclusion chromatography, native polyacrylamide and agarose gel electrophoresis. The data suggest that aggregates of four closely related tOx-PLs form micelle-like particles of considerably different properties. Our result provides first direct evidence that because of the specific chemical composition of the sn-2 fatty acyl chain aggregates of particular tOx-PL possess a distinctive set of physicochemical properties.     

Action of human lysosomal elastase on the oxidized B chain of insulin.

The specificity of action of the lysosomal elastase of human neutrophil leucocytes on the oxidized B chain of insulin is similar to that of pig pancreatic elastase, but is more directed towards valine than alanine as the residue contributing the carboxyl group of the cleaved bond. The most susceptible bonds are Val-12-Glu-13 and Val-18-Cys(O3H)-19. Other bonds hydrolysed are Ala-14-Leu-15, Ser-9-His-10 and Cys, (O3H3)-7-Gly-8. Tables listing amino acid composition, N-terminal residue, and yields of isolated peptides have been deposited as Supplementary Publication SUP 50 075 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161, 1.     

Action of human cathepsin G on the oxidized B chain of insulin.

The specificity of cathepsin G, a serine neutral proteinase from human neutrophil leucotyes, was determine dby its action on the insulin B chain. The most susceptible bonds were Phe-24-Phe-25, Leu-15-Tyr-16 and Tyr-16-Leu-17. Other bonds hydrolysed were Leu-6-Cys(O3H)-7, Leu-11-Val-12, Leu-17-Val-18 and Phe-25-Tyr-26. These results suggest that the specificity of cathespin G is closer to that of pig chymotrypsin C than ox Chymotrypsin A. Tables listing amino acid composition, N-terminal residue, and yields of isolated peptides have been deposited as Supplementary Publication SUP 50 075 (8 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7B2, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1977) 161,1.     

Action of human liver cathepsin B on the oxidized insulin B chain.

The lysosomal cysteine proteinase cathepsin B (from human liver) was tested for its peptide-bond specificity against the oxidized B-chain of insulin. Sixteen peptide degradation products were separated by high-pressure liquid chromatography and thin-layer chromatography and were analysed for their amino acid content and N-terminal amino acid residue. Five major and six minor cleavage sites were identified; the major cleavage sites were Gln(4)-His(5), Ser(9)-His(10), Glu(13)-Ala(14), Tyr(16)-Leu(17) and Gly(23)-Phe(24). The findings indicate that human cathepsin B has a broad specificity, with no clearly defined requirement for any particular amino acid residues in the vicinity of the cleavage sites. The enzyme did not display peptidyldipeptidase activity with this substrate, and showed a specificity different from those reported for two other cysteine proteinases, papain and rat cathepsin L.     

Adsorption of polypeptides on solid surfaces. I. Effect of chain stiffness

The general method of obtaining the partition function and thermodynamic characteristics of polymer chains near an adsorbing surface, simulated by random walks on a lattice, is developed. The method takes into account the effect of short-range interactions in polymer chains, in particular, the chain stiffness and secondary structure. The theory of adsorption of chains of different stiffness is developed, and the process of adsorption which occurs when the external conditions change is shown to be always a second-order phase transition. The critical adsorption energy decreases and the sharpness of transition grows when the chain stiffness increases. A simple model of a chain with “virtual” steps is proposed which simplifies the treatment; the results obtained are in good agreement with exact theories. A general scheme of analysis of adsorption of chains with a given secondary structure is set forth and the analogy between the stiffness of a noncooperative chain and the presence of helical segments in a polypeptide chain is discussed.     

Evaluation and characterization of EIA measuring autoantibodies against oxidized LDL

Autoantibodies against oxidized LDL (oxLDL) have been measured in many laboratories. Comparison of data between laboratories is difficult because of methodological variations and differences in the expression of results. We have optimized an enzyme immunoassay (EIA), which measures autoantibodies against oxLDL and evaluated the effect on results of different ways of expressing the data. Optimized conditions were as follows: coating concentration 2 microg/ml of LDL on polysorp plates, 1% human serum albumin (HSA) as a blocking agent, sample dilution 1:50, conjugate dilution 1:8000, and 0.2% HSA in sample and conjugate diluents. The amount of autoantibodies expressed as ratios between oxLDL and native LDL (natLDL), as titers against oxLDL or as differences between binding to oxLDL and natLDL showed significant differences among groups of coronary heart disease (CHD) patients with different diagnosis or treatment procedures. However, there were no differences among the groups when the results were expressed as the ratio between antibody titer against oxLDL and a standard serum (oxLDL/stand). After standardization oxLDL autoantibody test may become a useful tool for analysis of the risk for CHD.     

Denatured state thermodynamics: residual structure, chain stiffness and scaling factors

A set of nine variants of yeast iso-1-cytochrome c with zero or one surface histidine have been engineered such that the N-terminal amino group is acetylated in vivo. N-terminal acetylation has been confirmed by mass spectral analysis of intact and proteolytically digested protein. The histidine-heme loop-forming equilibrium, under denaturing conditions (3 M guanidine hydrochloride), has been measured by pH titration providing an observed pK(a), pK(a)(obs), for each variant. N-terminal acetylation prevents the N-terminal amino group-heme binding equilibrium from interfering with measurements of histidine-heme affinity. Significant deviation is observed from the linear dependence of pK(a)(obs) on the log of the number of monomers in the loop formed, expected for a random coil denatured state. The maximum histidine-heme affinity occurs for a loop size of 37 monomers. For loop sizes of 37-83 monomers, histidine-heme pK(a)(obs) values are consistent with a scaling factor of -4.2+/-0.3. This value is much larger than the scaling factor of -1.5 for a freely jointed random coil, which is commonly used to represent the conformational properties of protein denatured states. For loop sizes of nine to 22 monomers, chain stiffness is likely responsible for the decreases in histidine-heme affinity relative to a loop size of 37. The results are discussed in terms of residual structure and sequence composition effects on the conformational properties of the denatured states of proteins.