Pentacoordinate and hexacoordinate ferric hemes in acid medium: EPR, UV-Vis and CD studies of the giant extracellular hemoglobin of Glossoscolex paulistus

The equilibrium complexity involving different axially coordinated hemes is peculiar to hemoglobins. The pH dependence of the spontaneous exchange of ligands in the extracellular hemoglobin from Glossoscolex paulistus was studied using UV-Vis, EPR, and CD spectroscopies. This protein has a complex oligomeric assembly with molecular weight of 3.1 MDa that presents an important cooperative effect. A complex coexistence of different species was observed in almost all pH values, except pH 7.0, where just aquomet species is present. Four new species were formed and coexist with the aquomethemoglobin upon acidification: (i) a "pure" low-spin hemichrome (Type II), also called hemichrome B, with an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (ii) a strong g(max) hemichrome (Type I), also showing an usual spin state (d(xy))(2)(d(xz),d(yz))(3); (iii) a hemichrome with unusual spin state (d(xz),d(yz))(4)(d(xy))(1) (Type III); (iv) and a high-spin pentacoordinate species. CD measurements suggest that the mechanism of species formation could be related with an initial process of acid denaturation. However, it is worth mentioning that based on EPR the aquomet species remains even at acidic pH, indicating that the transitions are not complete. The "pure" low-spin hemichrome presents a parallel orientation of the imidazole ring planes but the strong g(max) hemichrome is a HALS (highly anisotropic low-spin) species indicating a reciprocally perpendicular orientation of the imidazole ring planes. The hemichromes and pentacoordinate formation mechanisms are discussed in detail.     

The interaction of hemoglobin with hexadecyltrimethylammonium bromide

The interaction of hemoglobin (Hb) with hexadecyltrimethylammonium bromide (CTAB) is investigated by UV–vis absorption spectra and fluorescence spectra method. CTAB monomer can convert methemoglobin (metHb) to hemichrome, and CTAB molecular assemblies, such as micelle, microemulsion and lamellar liquid crystal, can induce heme monomer to leave the hydrophobic cavity of Hb. TEM results show that Hb maintains the spherical structure in CTAB microemulsions while it is unfolded in CTAB lamellar liquid crystals. The existence of proton in the above systems can increase the stability of metHb.     

The interaction between nitrogen oxides and hemoglobin and endothelium-derived relaxing factor

Among nitrogen oxides, NO and NO₂ are free radicals and show a variety of biological effects. NO₂ is a strongly oxidizing toxicant, although NO, not oxidizing as NO₂, is toxic in that it interacts with hemoglobin to form nitrosyl-and methemoglobin. Nitrosylhemoglobin shows a characteristic electron spin resonance (ESR) signal due to an odd electron localized on the nitrogen atom of NO and reacts with oxygen to yield nitrate and methemoglobin, which is rapidly reduced by methemoglobin reductase in red cells. NO was found to inhibit the reductase activity. Part of NO inhaled in the body is oxidized by oxygen to NO₂, which easily dissolves in water and converts to nitrite. The nitrite oxyhemoglobin autocatalytically after a lag. The mechanism of the oxidation, particularly the involvement of superoxide, was controversial. The stoichiometry of the reaction has now been established using nitrate ion electrode and a methemoglobin free radical was detected by ESR during the oxidation. Complete inhibition of the autocatalysis by aniline or aminopyrine suggests that the radical catalyzes conversion of nitrite to NO₂, which oxidizes oxyhemoglobin. Recently NO was shown to be one of endothelium- derived relaxing factors and the relaxation induced by the factor was inhibited by hemoglobin and potentiated by superoxide dismutase.     

The interaction of hemoglobin with phosphatidylserine vesicles

The interaction of hemoglobin with phosphatidylserine vesicles at low ionic strength and pH conditions was studied. The fluorescence intensity of a lipid embedded probe was quenched by bound Hb but could not be reversed by an elevation of ionic strength and pH. The irreversibility of the fluorescence quenching is a time-dependent process associated with changes in the heme Soret and visible spectra. The rate of these changes was much faster for methemoglobin than for either cyanomethemoglobin or oxyhemoglobin. Elevation of ionic strength released out of the bound hemoglobin into the water phase most of the globin but only a small fraction of the heme. The data are interpreted as demonstrating the ability of phosphatidylserine vesicles to compete with globin for the heme group. When Hb binds to the liposome, heme is being transferred into the lipid phase and the rate-limiting step is the dissociation of the heme-globin complex. The fact that binding of heme to the lipid vesicles is very strong was demonstrated by the failure of hemin to interact with globin when the two were rapidly mixed in the presence of phosphatidylserine vesicles. A multi-step process is suggested to explain the results of Hb phosphatidylserine interaction.     

Interaction behaviors between chitosan and hemoglobin

The interaction and association between chitosan and hemoglobin (Hb) are studied by UV-vis and fluorescence spectroscopies, viscometry, circular dichroism, dynamic and static light scattering. Chitosan can obviously associate with Hb to form protein-chitosan complexes, which affects microstructure of Hb. The distance between the first association site of chitosan with 214-tryptophan residue in Hb is about 5.473 nm. The intrinsic UV-vis absorption and fluorescence intensities of Hb increase with an increase of chitosan concentration. The alpha-helix in Hb is drawn and changed into beta-sheet.     

Holo- and apo-cystalysin from Treponema denticola: two different conformations

Cystalysin, the key virulence factor in the bacterium Treponema denticola responsible for periodontis, is a pyridoxal 5′-phosphate (PLP) enzyme which catalyzes, in addition to α,β-elimination of l-cysteine, racemization and transamination of both enantiomers of alanine. In this paper several indicators have been used as probes of the different conformational status of T. denticola cystalysin in the holo and apo form. Compared to holoenzyme, the apoenzyme displays an altered reactivity of cysteine residues, a significantly different pI, and a differential susceptibility to proteinase K. The site of cleavage that is accessible in apocystalysin and masked in holocystalysin has been identified by mass spectrometry as the peptide bond between Phe 360 and Gly 361. This cleavage results in the loss of the C-terminal fragment corresponding to a molecular mass of 4289.21 ± 0.1 Da. The major fragment of cleaved enzyme retains its dimeric structure, binds the coenzyme with an affinity ∼5000-fold lower than that of uncleaved holoenzyme, and in the reconstituted form is able to form the external aldimine with substrates. Although the break causes the loss of lyase, racemase and transaminase activities of d-alanine, it does not abolish the transaminase activity of l-alanine. Possible mechanistic and physiological implications are proposed.     

The interaction between ribonuclease A and surfactants

1. U.v. difference spectra show that the anionic surfactant sodium n-dodecyl sulphate unfolds ribonuclease A at pH7.3 and 10.3, but that the cationic surfactant n-dodecyltrimethylammonium bromide does not affect the conformation of the enzyme. 2. Equilibrium-dialysis experiments show that sodium n-dodecyl sulphate binds to ribonuclease A, but no binding of n-dodecyltrimethylammonium bromide could be detected at pH7.3. 3. The enzymic activity of ribonuclease A is unaffected by n-dodecyltrimethylammonium bromide up to a concentration of 0.03m at 25 degrees C. 4. Ultracentrifuge studies support the conclusion that n-dodecyltrimethylammonium bromide does not interact significantly with ribonuclease A. 5. The enthalpy change as measured by microcalorimetry on binding of sodium n-dodecyl sulphate to ribonuclease A is consistent with an exothermic enthalpy of binding occurring simultaneously with an endothermic enthalpy of chain unfolding.     

Study of the interaction between DNP and DIDS with human hemoglobin as binary and ternary systems: spectroscopic and molecular modeling investigation

The combination of several drugs is necessary, especially during long-term therapy. A competitive binding of the drugs can cause a decrease in the amount of drugs actually bound to the protein and increase the biologically active fraction of the drug. Here, the interaction between 4,4′-Diisothiocyano-2,2′-stilbenedisulfonic acid (DIDS) and 2,4-Dinitrophenol (DNP) with Hemoglobin (Hb) was investigated by different spectroscopic and molecular modeling techniques. Fluorescence analysis was used to estimate the effect of the DIDS and DNP on Hb as well as to define the binding properties of binary and ternary complexes. The distance r between donor and acceptor was obtained by the FRET and found to be 2.25 and 2.13 nm for DIDS and DNP in binary and 2.08 and 2.07 nm for (Hb–DNP) DIDS and (Hb–DIDS) DNP complexes in ternary systems, respectively. Time-resolved fluorescence spectroscopy confirmed static quenching for Hb in the presence of DIDS and DNP in both systems. Furthermore, an increase in ellipticity values of Hb upon interaction with DIDS and DNP showed secondary structural changes of protein that determine to disrupt of hydrogen bonds and electrostatic interactions. Our results showed that the Hb destabilize in the presence of DIDS and DNP. Molecular modeling of the possible binding sites of DIDS and DNP in binary and ternary systems in Hb confirmed the experimental results.     

The interaction between bovine serum albumin and surfactants.

1. Potassium n-decyl phosphate binds exothermically to bovine serum albumin at pH 7.0 to form a specific complex containing approx. 60 phosphate anions. 2. The formation of the complex is accompanied by changes in the u.v. difference spectrum of the protein. 3. At higher phosphate concentrations (above 0.4mM) surfactant molecules continue to be bound, and the protein undergoes a gross change in conformation. 4. n-Dodecyltri-methylammonium bromide binds endothermically to bovine serum albumin at pH7.0 but the extent of binding for a given free surfactant concentration is less than for the phosphate surfactant. 5. Binding is accompanied by a small change in the specific viscosity and by changes in the u.v. difference spectrum of the protein. 6. It is suggested that over the surfactant concentration ranges studied n-decyl phosphate ions first bind to the C-terminal part of the protein and then to the more compact N-terminal part whereas n-dodecyltrimethylammonium ions bind only to the C-terminal part of bovine serum albumin.     

The partial sequence of the first 30 residues from the amino-terminus of hemoglobin B in the hagfish,Eptatretus stoutii: Homology with lamprey hemoglobin

Summary The partial sequence of the first 30 residues from the amino-terminus of hemoglobin B in the hagfish,Eptatretus stoutii, has been determined. Considerable homology was found between this sequence and the corresponding sequence of lamprey hemoglobin. Both hagfish and lamprey hemoglobins have an additional segment of 9 residues at the amino-terminus as compared with mammalian hemoglobins.This work was initiated at the University of Texas at Austin, and continued in Dr. Charles Yanofskey's laboratory at Stanford University.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Spontaneous formation of DPPC monolayers at aqueous/vapor interfaces and the impact of charged surfactants

Surface tensiometry and vibrational sum-frequency spectroscopy were used to examine the structure and organization in phospholipid monolayers at the aqueous/vapor interface in the absence and in the presence of simple, charged surfactants. 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) was the phospholipid employed in these studies and surfactants included sodium dodecyl sulfate (SDS) and dodecyl trimethyl ammonium bromide (DTAB). DPPC spontaneously spreads on a pure water (pH = 5.5) surface to form monolayers as evidenced by an equilibrium spreading pressure (ESP) of 7.9 ± 2.3 mN/m and a clearly resolved vibrational spectrum. Low concentrations of surfactants inhibit the spreading of DPPC and result in significantly lower ESP values. Anionic and cationic surfactants at higher concentrations have opposite effects on monolayer organization; SDS creates well-organized monolayers while DTAB leads to poor organization of lipid molecules. Surface-specific vibrational spectra showed that high concentrations of charged surfactants (≥ 100 µM) lead to accumulation of net surface charges as evidenced by destructive and constructive interferences. Selectively deuterating surfactants results in changes in vibrational band intensities and phases enabling assignment of relative orientations of equivalent functional groups belonging to the lipid and surfactant.     

Hemoglobin polymorphism in macaques with reference to the evolution ofMacaca fascicularis andMacaca mulatta

Blood samples were collected fromMacaca fascicularis andMacaca mulatta living in indoor breeding groups and investigated electrophoretically. Hemoglobin polymorphism was observed in both species. Isoelectric focusing was performed on urea denaturated samples to test the hypothesis of a site duplication at theα-chain locus inM. fascicularis (Barnicot et al., 1970). The results of our investigations do not support the above mentioned hypothesis. Only one locus coding theα-chain was detected, and this is under the control of two alleles. Evolutionary events at the molecular level are discussed, as well asWheatley's hypothesis (1980) that malaria was an important force behind divergence in both species. InM. fascicularis hemoglobin variants might be similarily connected with malaria resistance as in man. We suggest that this was not an important process behind speciation in macaques.     

Discrimination between closed and open forms of lipases using electrophoretic techniques

The enhanced catalytic activity of lipases is often associated with structural changes. The three-dimensional (3D) structures showed that the covalently inhibited lipases exist under their open conformations, in contrast to their native closed forms. We studied the inhibition of various lipases--human and dog gastric lipases, human pancreatic lipase, and Humicola lanuginosa lipase--by the octyl-undecyl phosphonate inhibitor, and we measured the subsequent modifications of their respective electrophoretic mobility. Furthermore, the experimental values of the isoelectric points found for the native (closed) and inhibited (open) lipases are in agreement with theoretical calculations based on the electrostatic potential. We concluded that there is a significant difference in the isoelectric points between the closed (native) and open (inhibited) conformations of the four lipases investigated. Thus, analysis of the electrophoretic pattern is proposed as an easy experimental tool to differentiate between a closed and an open form of a given lipase.     

The model of Monod, Wyman, and Changeux generates two sets of parameters when applied to oxygen binding in hemoglobin

The model of Monod, Wyman and Changeux is applied to binding phenomena where the Mass Law and its expansion are employed. In this communication the model of Monod, Wyman and Changeux (MWC) is applied to analyze the oxygen binding reaction in hemoglobin. The symmetrical structure of the MWC model with its three parameters is such that two sets of these parameters, rather than one, fit experimental data for the binding of oxygen to hemoglobin.     

The interaction between heparin and antithrombin III: a comparison of two different heparin-dye conjugates

The interactions of antithrombin III with two heparin-dye conjugates have been compared using their fluorescence anisotropy. The first, heparin labelled with 5-isothiocyanatofluorescein, where the dye was mostly bound to unsulphated glucosamine residues, exhibited binding which was characteristic of heparin with a low affinity for antithrombin III. The second, heparin labelled with a reactive naphthalene dye (DENMT), showed similar binding character. However, when the heparin was treated with an amino group blocking agent prior to labelling with DENMT, the resultant heparin-dye conjugate showed binding behaviour, the strength of which was consistent with heparin molecules having both high and low affinity for antithrombin III. Heparin molecules with a high affinity for antithrombin III did not possess free amino groups. The implications of these findings are discussed with regard to the reliability of the data obtained using heparin-fluorescein conjugates.     

The interaction of chloride ions with human hemoglobin

The immobilization of glucose oxidase, a glycoenzyme from $\text{Aspergillus}$$\text{niger}$ consisting of 16% carbohydrate, has been achieved by oxidizing its carbohydrate residues with periodic acid followed by coupling the activated enzyme to water-insoluble $\text{p}$-aminostyrene. At pH 5.6 and 25°, approximately 60% of the carbohydrate residues are oxidized, but the enzyme retains full activity. No oxidation of any amino acid residue is evident. The enzyme-polymer conjugate derived from this activated enzyme retains full activity and even shows a slightly enhanced thermal stability at 60° compared with the soluble native and oxidized glucose oxidases.     

Characterization of the interaction between surfactants and enzymes by fluorescence probe

In order to investigate the mechanism of the different stimulatory effects of the biosurfactant rhamnolipid and the chemical surfactant Tween 80 on enzymatic hydrolysis of lignocellulose, the interaction between surfactants and enzymes was analyzed by the fluorescence probe method using pyrene as probe. Based on the evolution law of pyrene fluorescence spectroscopy in the “surfactants-enzymes” systems, the interaction relationship between surfactants and enzymes was analyzed and discussed in this paper. The results show that enzyme molecules bind with rhamnolipid molecules, participate in the formation of rhamnolipid micelles, and increase the inner hydrophobic polarity of micelles, but do not change the properties of rhamnolipid micelles above the CMC (Critical Micelle Concentration). Nevertheless, for Tween 80, enzyme molecules also participate in the forming of micelles, however, they exhibit a stronger interaction with enzymes above the CMC. Both rhamnolipid and Tween 80 bind more strongly with xylanase than cellulase. Considering also previous experimental results, it can be concluded that the interaction between surfactants and enzymes improve enzyme stability and activity, and, therefore, the efficiency of enzymatic hydrolysis of lignocellulose is enhanced. The findings further provide theoretical knowledge about the mechanism of the stimulative effects of surfactants on enzymatic hydrolysis of lignocellulose.