Mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid to bovine serum albumin

The mechanism of the binding of 2-(4'-hydroxyphenylazo)benzoic acid (HABA) to bovine serum albumin was studied by relaxation methods as well as the binding isotherm using gel chromatography. A single relaxation was observed over a wide range of HABA concentration except at the extremes of high concentration where another slow process was observed. The concentration dependence of the reciprocal relaxation time of the fast process decreased monotonically with increase in concentration of HABA at constant polymer concentration. The data were analyzed on the basis of Brown's domain structure model and were found to be consistent with a sequential binding mechanism. The azohydrazon tautomerism of HABA was identified with the intramolecular step of the complex. The activation parameters of the step, determined from the temperature dependence of the relaxation time of the fast process, showed that this step is rate limited by an enthalpy barrier in both forward and backward directions. Comparison of the activation parameters with those of other serum albumin-ligand systems suggests that there is an enthalpy-entropy compensation in the activation process of the intramolecular step with the compensation temperature at about 270 K; the enthalpy-entropy compensation is thought to be related to the hydrophobic nature of the ligand.     

Influence of Nutrient Concentrations on MPN Quantification and Enrichment of Nitrate-Reducing Fe(II)-Oxidizing and Fe(III)-Reducing Bacteria from Littoral Freshwater Lake Sediments

The application of culture-dependent studies to quantify Fe-metabolizing microorganisms from the environment is a necessity, as there are so far no universal functional marker genes for application in culture-independent studies. Media composition can vary between studies, therefore, we determined the effects of three different growth media on the quantification (MPNs) and identity (via cloning and sequencing of dominant DGGE bands) of nitrate-reducing Fe(II)-oxidizers and lactate- or acetate-oxidizing Fe(III)-reducers from a lacustrine sediment: low sulphate freshwater medium (FWM), sterile filtered bicarbonate-buffered lake water (BLW) and a mixture of both (MIX). We consistently found fewer cells in the BLW than in the FWM and the MIX. The DGGE banding patterns of the microbial communities enriched in different media types clustered together according to the e^? donor and acceptor couples and not according to the medium used. Thus, although the medium composition significantly influenced the quantification and thereby conclusions on the abundance and potential significance of the targeted group within the ecosystem, biodiversity assessments through enrichment cultures were less influenced by the medium, but instead were affected by the type and concentration of the e^? donor/acceptor.     

Iron uptake byBifidobacterium thermophilum protoplasts

Protoplasts ofBifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO₄. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.     

Iron supplementation moderates but does not cure the Belgrade anemia

Belgrade rats inherit microcytic, hypochromic anemia as an autosomalrecessive trait (gene symbol b). Erythrocytes and tissue are iron deficientin the face of elevated TIBC (total iron binding capacity) and percent ironsaturation; iron injections increased the number of erythrocytes but theirappearance remained abnormal. We have investigated iron supplements toimprove husbandry of b/b rats and to learn more about the underlying defectand its tissue distribution. Weekly IM (intramuscular) injections ofiron–dextran (Imferon at 30 mg kg) improved the anemia but did not alter thered cell morphology. Certain diets also improved the health of b/b rats whencompared to standard rat chows by the criteria of weight, survival toadulthood, hematology and reproduction. The critical nutritional factorturned out to be iron bioavailability, with ferrous iron added to the dietimproving the health of Belgrade rats without affecting the underlyingerythroid defect. Tissue iron measurements after dietary or parenteralsupplementation confirmed the iron deficient status of untreated b/b rats andestablished that dietary ferrous iron partially relieved this deficiency,with injections leading to greater amounts of tissue iron. Serum iron andTIBC were also found to be elevated in untreated b/b rats, with dietarysupplementation decreasing but not eliminating the elevation in TIBC. Thesestudies indicate that iron supplements can improve the health of b/b ratswithout altering the underlying defect and also suggest that the mutationcould alter iron uptake in the GI (gastrointestinal) tract.     

Circular dichroism of ketoprofen complexed to serum albumins: Conformational selection by the protein: A novel optical purity determination technique

Complexation of 2-(3′-benzoylphenyl)propionic acid (ketoprofen), 1 , to bovine serum albumin (BSA) results in an intense negative circular dichroism in the ketonic n → π* band of the benzoylphenyl moiety. This high CD contrasts with the weak CD of 1 -enantiomers dissolved in common solvents. Furthermore, a number of chiral and achiral molecules containing the benzophenone moiety are easily complexed to BSA: all these complexes show an intense CD at the same transition. To account for the observed CD intensities of the above molecules, it appears that BSA complexation markedly shifts the equilibrium between strongly asymmetric, antipodic conformers. Dissymmetry of these conformers is connected to the instability of a structure with phenyl rings coplanar to the carbonyl chromophore, as also indicated by molecular mechanics calculations. The magnification of the Cotton effects of the 1 -antipodes, due to the protein, can be used to measure the optical purity of 1 -samples with excellent precision. In contrast with BSA, human SA is unable to recognize the chirality of 1 -antipodes; oleic acid cocomplexation modifies this fact as well as other features of the binding. © 1995 Wiley-Liss, Inc.     

Effect of ligand binding and conformational changes in proteins on oxygen quenching and fluorescence depolarization of tryptophan residues

The rotational freedom of tryptophan residues in protein-ligand complexes was studied by measuring steady-state fluorescence anisotropies under conditions of oxygen quenching. There was a decrease in the oxygen bimolecular quenching constant upon complexation of trypsin and alpha-chymotrypsin with proteinaceous trypsin inhibitors, of lysozyme with N-acetylglucosamine (NAG) and di(N-acetyl-D-glucosamine) ((NAG)2) and of hexokinase with glucose. Binding of the bisubstrate analogue N-phosphonacetyl-L-aspartate (PALA) to aspartate transcarbamylase (ATCase) and binding of biotin to avidin resulted in increased oxygen quenching constants. The tryptophan of human serum albumin (HSA) in the F state was more accessible to oxygen quenching than that in the N state. With the exception of ATCase, the presence of subnanosecond motions of the tryptophan residues in all the proteins is suggested by the short apparent correlation times for fluorescence depolarization and by the low apparent anisotropies obtained by extrapolation to a lifetime of zero. Complex formation evidently resulted in more rigid structures in the case of trypsin, alpha-chymotrypsin and lysozyme. The effects of glucose binding on hexokinase were not significant. Binding of biotin to avidin resulted in a shorter correlation time for the tryptophan residues. The N --> F transition in HSA resulted in a more rigid environment for the tryptophan residue. Overall, these changes in the dynamics of the protein matrix and motional freedom of tryptophan residues due to complex formation and subsequent conformational changes are in the same direction as those observed by other techniques, especially hydrogen exchange. Significantly, the effects of complex formation on protein dynamics are variable. Among the limited number of cases we examined, the effects of complex formation were to increase, decrease or leave unchanged the apparent dynamics of the protein matrix.     

Electron transfer between horse heart and Candida krusei cytochromes c in the free and bound states

Electron transfer between horse heart and Candida krusei cytochromes c in the free and phosvitin-bound states was examined by difference spectrum and stopped-flow methods. The difference spectra in the wavelength range of 540–560 nm demonstrated that electrons are exchangeable between the cytochromes c of the two species. The equilibrium constants of the electron transfer reaction for the free and phosvitin-bound forms, estimated from these difference spectra, were close to unity at 20°C in 20 mM Tris-HCl buffer (pH 7.4). The electron transfer rate for free cytochrome c was (2–3) · 10⁴ M^?1 · s^?1 under the same conditions. The transfer rate for the bound form increased with increase in the binding ratio at ratios below half the maximum, and was almost constant at higher ratios up to the maximum. The maximum electron exchange rate was about 2 · 10⁶ M^?1 · s^?1, which is 60–70 times that for the free form at a given concentration of cytochrome c. The activation energy of the reaction for the bound cytochrome c was equal to that for the free form, being about 10 kcal/mol. The dependence of the exchange rate on temperature, cytochrome c concentration and solvent viscosity suggests that enhancement of the electron transfer rate between cytochromes c on binding to phosvitin is due to increase in the collision frequency between cytochromes c concentrated on the phosvitin molecule.     

Studies of copper(II) binding to glycylglycyl-L-tyrosine-N-methyl amide, a peptide mimicking the NH2-terminal copper(II)-binding site of dog serum albumin by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy

Unlike human serum albumin (HSA), dog serum albumin (DSA) does not possess the characteristics of the specific first binding site for Cu(II). In DSA, the important histidine residue in the third position, responsible for the Cu(II)-binding specificity in HSA, is replaced by a tyrosine residue. In order to study the influence of the tyrosine residue in the third position of DSA, a simple model of the NH2-terminal native sequence tripeptide of DSA, glycylglycyl-L-tyrosine-N-methylamide (GGTNMA) was synthesized and its Cu(II)-binding properties studied by analytical potentiometry, spectrophotometry, CD, and NMR spectroscopy. The species analysis indicated the existence of five mono-complexes at different protonation states: MHA, MA, MH-1A, MH-2A, MH-3A, and only one bis-complex MH-2A-2. The complexing ability of GGTNMA to Cu(II) was found to be weaker than that of the Cu(II) binding peptide models of HSA. The visible absorption spectra of Cu(II)-GGTNMA complexes are similar to those observed in the case of DSA-Cu(II) complexes. The weaker binding and the spectral properties of Cu(II)-GGTNMA complexes are consistent with less specific Cu(II)-binding properties of the peptide of this sequence similar to what was noted with DSA. CD results are in excellent agreement with species analysis and visible spectra where it is clearly evident that Cu(II) binds to GGTNMA starting from the alpha-NH2 group and step by step to deprotonated amide nitrogens as the pH is raised. The absence of any charge transfer band around 400 nm strongly indicates that Cu(II) does not bind to the phenolate group. Furthermore, NMR results are consistent with the noninvolvement of the tyrosine residue of GGTNMA in Cu(II) complexation. Thus, it is clear that the low Cu(II)-binding affinity of DSA is due to the genetic substitution of tyrosine for histidine at the NH2-terminal region of the protein.     

Reactions of Adriamycin with Microsomal Iron and Lipids

Iron plays a central role in oxidative injury, reportedly because it catalyzes superoxide- and hydrogen peroxide-dependent reactions yielding a powerful oxidant such as the hydroxyl radical. Iron is also thought to mediate the cardiotoxic and antitumour effects of adriamycin and related compounds. NADPH-supplemented microsomes reduce adriamycin to a semiquinone radical, which in turn re-oxidizes in the presence of oxygen to form superoxide and hence hydrogen peroxide. During this redox cycling membrane-bound nonheme iron undergoes superoxide dismutase- and catalase-insensitive reductive release. Membrane iron mobilization triggers lipid peroxidation, which is markedly enhanced by simultaneous addition of superoxide dismutase and catalase. The results indicate that : i) lipid peroxidation is mediated by the release of iron, yet the two reactions are governed by different mechanisms; and ii) oxygen radicals are not involved in or may actually inhibit adriamycin-induced lipid peroxidation. Microsomal iron delocalization and lipid peroxidation might represent oxyradical-independent mechanisms of adriamycin toxicity.