Cyclic AMP-mediated intersubunit disulfide crosslinking of the cyclic AMP receptor protein of Escherichia coli.

The protomeric form of the cyclic AMP receptor protein (CRP) of Escherichia coli is composed of two identical subunits of molecular weight 22,500 and contains two buried and two available cysteine residues. Titration of the two available cysteines with DTNB⁴ eliminates cyclic AMP-dependent DNA binding activity which is regenerated by incubating the modified protein with β-mercaptoethanol. In the absence of cAMP, the formation of the TNB anion from DTNB and the incorporation of [¹⁴C]TNB into CRP are approximately stoichiometric. In the presence of cAMP, there is an increase in the rate of formation of the TNB anion while the incorporation of [¹⁴C]TNB into CRP is markedly inhibited. These observations are reconciled by the observation that cAMP induces DTNB-mediated disulfide crosslinking of the two available sulfhydryls to produce a species migrating as a 45,000 molecular weight subunit on SDS-polyacrylamide gels. A mechanism is suggested by which an intersubunit, intraprotomer disulfide bond is produced by secondary disulfide interchange after the incorporation of the initial TNB group. Based on the observation of cAMP-mediated disulfide crosslinking, the available cysteines of the DNA binding region are proposed to reside in close proximity as part of an antiparallel β-sheet structure formed by the two carboxyl proximal polypeptides when CRP is in the DNA binding conformation.     

Differences in coordination states of substituted tyrosine residues and quaternary structures among hemoglobin M probed by resonance Raman spectroscopy

Among the four types of hemoglobin (Hb) M with a substitution of a tyrosine (Tyr) for either the proximal (F8) or distal (E7) histidine in the α or β subunits, only Hb M Saskatoon (βE7Tyr) assumes a hexacoordinate structure and its abnormal subunits can be reduced readily by methemoglobin (metHb) reductase. This is distinct from the other three M Hbs. To gain new insight into the cause of the difference, we examined the ionization states of E7 and F8 Tyrs by UV resonance Raman (RR) spectroscopy and Fe–O(Tyr) bonding by visible RR spectroscopy. Hb M Iwate (αF8Tyr), Hb M Boston (αE7Tyr), and Hb M Hyde Park (βF8Tyr) exhibited two extra UV RR bands at 1,603 cm⁻¹ (Y8a′) and 1,167 cm⁻¹ (Y9a′) arising from deprotonated (ionized) Tyr, but Hb M Saskatoon displayed the UV RR bands of protonated (unionized) Tyr at 1,620 and 1,175 cm⁻¹ in addition to those of deprotonated Tyr. Evidence for the bonding of both ionization states of Tyr to the heme in Hb M Saskatoon was provided by visible RR spectroscopy. These results indicate that βE7Tyr of Hb M Saskatoon is in equilibrium between protonated and deprotonated forms, which is responsible for facile reducibility. Comparison of the UV RR spectral features of metHb M with that of metHb A has revealed that metHb M Saskatoon and metHb M Hyde Park are in the R (relaxed) structure, similar to that of metHb A, whereas metHb M Iwate, metHb M Boston and metHb M Milwaukee are in the T (tense) quaternary structure.     

Methemoglobin formation in mutant hemoglobin α chains: electron transfer parameters and rates

Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.     

Lanthanide ions induce hydrolysis of hemoglobin-bound 2,3-diphosphoglycerate (2,3-DPG), conformational changes of globin and bidirectional changes of 2,3-DPG-hemoglobin’s oxygen affinity

The changes in structure and function of 2,3-diphosphoglycerate-hemoglobin (2,3-DPG-Hb) induced by Ln³⁺ binding were studied by spectroscopic methods. The binding of lanthanide cations to 2,3-DPG is prior to that to Hb. Ln³⁺ binding causes the hydrolysis of either one from the two phosphomonoester bonds in 2,3-DPG non-specifically. The results using the ultrafiltration method indicate that Ln³⁺ binding sites for Hb can be classified into three categories: i.e. positive cooperative sites (N_I), non-cooperative strong sites (N_S) and non-cooperative weak sites (N_W) with binding constants in decreasing order: K_I>K_S>K_W. The total number of binding sites amounts to about 65 per Hb tetramer. Information on reaction kinetics was obtained from the change of intrinsic fluorescence in Hb monitored by stopped-flow fluorometry. Fluctuation of fluorescence dependent on Ln³⁺ concentration and temperature was observed and can be attributed to the successive conformational changes induced by Ln³⁺ binding. The results also reveal the bidirectional changes of the oxygen affinity of Hb in the dependence on Ln³⁺ concentration. At the range of [Ln³⁺]/[Hb]<2, the marked increase of oxygen affinity (P₅₀ decrease) with the Ln³⁺ concentration can be attributed to the hydrolysis of 2,3-DPG, while the slight rebound of oxygen affinity in higher Ln³⁺ concentration can be interpreted by the transition to the T-state of the Hb tetramer induced by Ln³⁺ binding. This was indicated by the changes in secondary structure characterized by the decrease of α-helix content.     

Quaternary structures and low frequency molecular vibrations of haems of deoxy and oxyhaemoglobin studied by resonance Raman scattering

The influence of quaternary structure on the low frequency molecular vibrations of the haem within deoxyhaemoglobin (deoxy Hb) and Oxyhaemoglobin (oxy Hb) was studied by resonance Raman scattering. The FeO₂ stretching frequency was essentially identical between the high affinity (R) state (Hb A) and low affinity (T) state (Hb Kansas and Hb M Milwaukee with inositol hexaphosphate). However in deoxy Hb, only one of the polarized lines showed an appreciable frequency shift upon switch of quaternary structure, i.e. 215 to 218 cm^?1 for the T state (Hb A, des-His(146β) Hb, and des-Arg(141α) Hb (pH 6.5)) and 220 to 221 cm^?1 for the R state (des-Arg(141α) Hb (pH 9.0), des-His(146β)-Arg(141α) Hb and NES des-Arg(141α) Hb). Based on the observed ⁵⁴Fe isotopic frequency shift of the corresponding Raman lines of deoxy Hb A (214 → 217 cm^?1), of deoxy NES des-Arg Hb (220 → 223 cm^?1), of the protoporphyrinato-Fe(II)-(2-methylimidazole) complex in the ferrous high spin state (207 → 211 cm^?1) and of deoxymyoglobin (220 → 222 cm^?1) (Kitagawa et al., 1979), and on substitution of perdeuterated for protonated 2-methylimidazole in the deoxygenated picket fence complex (T_pivPP)Fe²⁺ (2-MeIm) (209 → 206 cm^?1), and on the results of normal co-ordinates calculation carried out previously, we proposed that the 216 cm^?1 line of deoxy Hb is associated primarily with the FeN_ε(HisF8) stretching mode and accordingly that the FeN_ε(HisF8) bond is stretched in the T state due to a strain exerted by globin.     

Serine 312 phosphorylation is dispensable for wild-type p53 functions in vivo

Cellular stimulation results in phosphorylation of the tumor suppressor p53 on multiple residues, though the functional relevance is not always clear. It is noteworthy that the serine (S) 315 residue is unique, as it has been suggested to be phosphorylated not only by genotoxic signals, but also during cell-cycle progression and by endoplasmic-reticulum stress. However, in vitro data have been conflicting as phosphorylation at this site was shown to both positively and negatively regulate p53 functions. We have thus generated knock-in mice expressing an unphosphorylable S312 (equivalent to human S315), by substitution with an alanine (A) residue, to clarify the conflicting observations and to evaluate its functional relevance in vivo. Born at Mendelian ratios, the p53^S312A/S312A mice show no anomalies during development and adulthood. p53 activation, stability, localization and ability to induce apoptosis, cell-cycle arrest and prevent centrosome amplification are not compromised in p53^S312A/S312A cells. p53^S312A/S312A mice are unable to rescue mdm2^−/− lethality, and tumorigenesis – both spontaneous and irradiation/oncogene-induced – is not accentuated. Taken together, the results show that the S312 phosphorylation site is not in itself necessary for efficient p53 function, and advocates the possibility that it is neither relevant in the mouse context nor important for p53 functions in vivo.     

Hemoglobins Austin and Waco: two hemoglobins with substitutions in the alpha 1 beta 2 contact region.

Hemoglobins (Hbs) Austin and Waco were detected by their electrophoretic migration on cellulose acetate (pH 8.4) and citrate agar (pH 6.2). By these methods, both variants migrated between Hbs A and F. Globin chain analysis at pH 8.6 indicated that the mutant β chain of Hb Austin was faster moving than the β^A chain; however, the mutant chain of Hb Waco was indistinguishable from the β^A chain by this technique. The two variants were isolated by ion-exchange column chromatography. Sequence studies demonstrated a substitution of serine (Hb Austin) and lysine (Hb Waco) for arginine at position 40 in the β chain. These mutations involve an amino acid residue in the α₁β₂ contact region, which, before this report, had been considered invariant in all hemoglobin sequences. Hb Austin was found to exist as dimers when oxygenated and as tetramers when deoxygenated. The equilibrium constant (K_d) for the tetramer to dimer transition was approximately 300 × 10^?6m, as calculated from sedimentation velocity studies. This variant also had high oxygen affinity, a much reduced heme-heme interaction, and a normal Bohr effect. The functional properties of Hb Waco were similar to those of Hb A.     

Detection of interaction between lysionotin and bovine serum albumin using spectroscopic techniques combined with molecular modeling

A combination of fluorescence, UV–Vis absorption, circular dichroism (CD), Fourier transform infrared (FT-IR) and molecular modeling approaches were employed to determine the interaction between lysionotin and bovine serum albumin (BSA) at physiological pH. The fluorescence titration suggested that the fluorescence quenching of BSA by lysionotin was a static procedure. The binding constant at 298 K was in the order of 10⁵ L mol^?1, indicating that a high affinity existed between lysionotin and BSA. The thermodynamic parameters obtained at different temperatures (292, 298, 304 and 310 K) showed that the binding process was primarily driven by hydrogen bond and van der Waals forces, as the values of the enthalpy change (ΔH°) and entropy change (ΔS°) were found to be ?40.81 ± 0.08 kJ mol^?1 and ?35.93 ± 0.27 J mol^?1 K^?1, respectively. The surface hydrophobicity of BSA increased upon interaction with lysionotin. The site markers competitive experiments revealed that the binding site of lysionotin was in the sub-domain IIA (site I) of BSA. Furthermore, the molecular docking results corroborated the binding site and clarified the specific binding mode. The results of UV–Vis absorption, CD and FT-IR spectra demonstrated that the secondary structure of BSA was altered in the presence of lysionotin.     

Methemoglobin formation in mutant hemoglobin α chains: electron transfer parameters and rates

Hemoglobin-mediated transport of dioxygen (O₂) critically depends on the stability of the reduced (Fe²⁺) form of the heme cofactors. Some protein mutations stabilize the oxidized (Fe³⁺) state (methemoglobin, Hb M), causing methemoglobinemia, and can be lethal above 30%. The majority of the analyses of factors influencing Hb oxidation are retrospective and give insights only for inner-sphere mutations of heme (His58, His87). Herein, we report the first all-atom molecular dynamics simulations on both redox states and calculations of the Marcus electron transfer (ET) parameters for the α chain Hb oxidation and reduction rates for Hb M. The Hb wild-type (WT) and most of the studied α chain variants maintain globin structure except the Hb M Iwate (H87Y). The mutants forming Hb M tend to have lower redox potentials and thus stabilize the oxidized (Fe³⁺) state (in particular, the Hb Miyagi variant with K61E mutation). Solvent reorganization (λ_solv 73–96%) makes major contributions to reorganization free energy, whereas protein reorganization (λ_prot) accounts for 27–30% except for the Miyagi and J-Buda variants (λ_prot ∼4%). Analysis of heme-solvent H-bonding interactions among variants provide insights into the role of Lys61 residue in stabilizing the Fe²⁺ state. Semiclassical Marcus ET theory-based calculations predict experimental k_ET for the Cyt b5-Hb complex and provide insights into relative reduction rates for Hb M in Hb variants. Thus, our methodology provides a rationale for the effect of mutations on the structure, stability, and Hb oxidation reduction rates and has potential for identification of mutations that result in methemoglobinemia.     

Direct electron transfer and bioelectrocatalysis of hemoglobin at a carbon nanotube electrode

A stable suspension of carbon nanotube (CNT) can be obtained by dispersing the CNT in the solution of the surfactant cetyltrimethylammonium bromide. CNT has promotion effects on the direct electron transfer of hemoglobin (Hb), which was immobilized onto the surface of CNT. The direct electron transfer rate of Hb was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of Hb, with the formal potential (E^0′) at about −0.343 V (vs. saturated calomel electrode) in the phosphate buffer solution (pH 6.8). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k_s) and the value of formal potential (E^0′) were estimated. The dependence of E^0′ on solution pH indicated that the direct electron transfer reaction of Hb is a one-electron transfer coupled with a one-proton transfer reaction process. The experimental results also demonstrated that the immobilized Hb retained its bioelectrocatalytic activity to the reduction of H₂O₂. The electrocatalytic current was proportional to the concentration of H₂O₂ at least up to 20 mM.     

Structure of deoxy hemoglobin C (beta six Glu replaced by Lys) in two crystal forms

Five crystal forms of the abnormal human hemoglobin Hb³ C (beta six Glu → Lys) have been grown. Two of them are grown with liganded Hb C, three with deoxy Hb C. The structures of two of the deoxy crystal forms were determined by the method of molecular replacement, using deoxy Hb A as the model structure. Fourier maps were calculated for each Hb C structure, using data to a resolution of 5 Å in one case and 4 Å in the second case. The structural differences between each deoxy Hb C structure and the deoxy Hb A model are found mostly at the molecular surface. Energetically favorable interactions involving the variant residue, beta six lysine, occur in both Hb C crystal forms, and could explain the lowered solubility and enhanced tendency of deoxy Hb C to crystallize in vivo.     

Rational design of pyridyl derivatives of vanillin for the treatment of sickle cell disease

Hypoxia-induced polymerization of sickle hemoglobin (Hb S) is the principal phenomenon that underlays the pathophysiology and morbidity associated with sickle cell disease (SCD). Opportunely, as an allosteric protein, hemoglobin (Hb) serves as a convenient and potentially critical druggable target. Consequently, molecules that prevent Hb S polymerization (Hb modifiers), and the associated erythrocyte sickling have been investigated–and retain significant interest–as a viable therapeutic strategy for SCD. This group of molecules, including aromatic aldehydes, form high oxygen affinity Schiff-base adducts with Hb S, which are resistant to polymerization. Here, we report the design and synthesis of novel potent antisickling agents (SAJ-009, SAJ-310 and SAJ-270) based on the pharmacophore of vanillin and INN-312, a previously reported pyridyl derivative of vanillin. These novel derivatives exhibited superior in vitro binding and pharmacokinetic properties compared to vanillin, which translated into significantly enhanced allosteric and antisickling properties. Crystal structure studies of liganded Hb in the R2 quaternary state in complex with SAJ-310 provided important insights into the allosteric and antisickling properties of this group of compounds. While these derivatives generally show similar in vitro biological potency, significant structure-dependent differences in their biochemical profiles would help predict the most promising candidates for successful in vivo pre-clinical translational studies and inform further structural modifications to improve on their pharmacologic properties.     

Structural and functional studies of Hemoglobin Wayne: An elongated α-chain variant

Hemoglobin Wayne (Hb Wayne) is a frame-shift, elongated α-chain variant that exists in two forms, with either asparagine or aspartic acid as residue 139. Oxygen equilibrium studies showed that stripped Hb Wayne Asn and Hb Wayne Asp possessed high oxygen affinity ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 0.60}$ and 0.23 mmHg at pH 7, respectively), were non-co-operative and have a markedly reduced Bohr effect ($\text{?Δ}\text{log}\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{/}\text{pH}\text{(7}\text{to}\text{8) = 0.34}$ and 0.10, respectively). Adding organic phosphate results in a decreased oxygen affinity and increased Bohr effect for both Hbs Wayne. The overall rate of carbon monoxide binding at pH 7 (l′ = 5.6 × 10⁶m^?1s^?1) was similar for both stripped Hbs Wayne and was 25-fold more rapid than that of stripped Hb A. When organic phosphate was added, Hb Wayne Asn exhibited a homogeneous slower rate of carbon monoxide binding (l′ = 2.6 × 10⁶m^?1s^?1), whereas Hb Wayne Asp showed heterogeneous binding (l′ = 6.1 × 10⁶ and 2.6 × 10⁶m^?1s^?1 for fast and slow phases, respectively). The rates of overall oxygen dissociation and oxygen dissociation with carbon monoxide replacement for both Hbs Wayne were found to be slow compared to Hb A and uniquely different from each other. Similarly, sedimentation velocity experiments indicated that, although Hb Wayne Asn and Hb Wayne Asp were both less tetrameric than Hb A, each hemoglobin exhibited a distinct degree of oxygen-linked subunit dissociation. These observed differences in the allosteric properties of Hb Wayne Asn and Hb Wayne Asp appeared to be directly attributable to residue 139. The equilibrium and kinetic data are consistent with the X-ray diffraction analysis of Hb Wayne Asp, which shows that the C terminus of the deoxytetramers are severely disordered, a condition that results in major destabilization of the T conformation and disruption of normal hemoglobin function.     

Study on the antibacterial activity and interaction with human serum albumin of Tagetes erecta inspired biogenic silver nanoparticles

Here, Tagetes erecta leaves extract mediated silver nanopartices (te-SNPs) were synthesized. This synthesis process was simple, one step and eco-friendly. The te-SNPs were characterized by various spectroscopic instruments such as ultraviolet visible (UV–vis), Fourier-transform infrared (FTIR) spectroscopy, dynamic light scattering (DLS) and Transmission Electron Microscopy (TEM) with energy Dispersive X-ray Spectroscopy (EDS). These te-SNPs showed the effective bactericidal activity against Escherichia coli and Staphylococcus aureus. The interaction of te-SNPs with human serum albumin (HSA) was evaluated by UV–vis absorption, fluorescence (FL), time resolved fluorescence (TRF), DLS and circular dichroism (CD) study. TRF and temperature dependant study suggested that the interaction process followed the dynamic quenching mechanism. The stern-volmer quenching constants (K_SV) were obtained as 1.36 × 10⁷, 1.51 × 10⁷ and 1.94 × 10⁷ M⁻¹at 288, 298 and 308 K respectively. The thermodynamic parameters were also evaluated and this result suggested that the interaction was spontaneous, and the hydrophobic forces played the most important role for the interaction process. In addition, CD spectra proved minute alteration of secondary structure of HSA upon interaction with the te-SNPs.     

加速康复外科对腹腔镜胃癌根治术患者营养状态,免疫功能及炎性因子水平的影响

目的:探讨加速康复外科(ERAS)对腹腔镜胃癌根治术患者营养状态,免疫功能及炎性因子水平的影响。方法:将2016年1月~2020年1月于我院行腹腔镜胃癌根治术的胃癌患者166例纳入本研究,按照随机数字表法分为ERAS组(n=83)与对照组(n=83),对照组行常规处理,ERAS组以ERAS处理。观察两组术后1 d、7 d血红蛋白(Hb)、视黄醇结合蛋白(RbP)、转铁蛋白(TRF)、前白蛋白(PRE)、白蛋白(ALB)等营养指标,免疫球蛋白A(Ig A)、免疫球蛋白G(Ig G)、免疫球蛋白M(Ig M)等免疫球蛋白,CD3⁺、CD4⁺、CD8⁺、CD4⁺/CD8⁺等T细胞亚群,白介素-6(IL-6)、白介素-8(IL-8)、白介素-10(IL-10)等炎性因子及术后并发症等指标。结果:与术后1 d比较,两组术后7 d Hb、RbP、TRF、PRE、ALB、Ig A、Ig G、Ig M,CD3⁺、CD4⁺、CD4⁺/CD8⁺水平均升高,CD8⁺、IL-6、IL-8、IL-10水平均降低(P<0.05)。术后7 d,ERAS组Hb、RbP、TRF、PRE、ALB、Ig A、Ig G、Ig M、CD3⁺、CD4⁺、CD4⁺/CD8⁺水平均高于对照组,CD8⁺、IL-6、IL-8、IL-10低于对照组(P<0.05)。ERAS组术后并发症发病率(8.43%)低于对照组(22.89%),差异有统计学意义(P<0.05)。结论:ERAS应用于腹腔镜胃癌根治术中可有效降低患者机体应激反应,改善营养状态,解除免疫抑制,清除炎性因子,减少术后并发症,有助于患者康复。     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: III. The influence of heme removal

Two main types of conformational fluctuations, local and global, are characteristic of the native protein structure and are detectable by hydrogen exchange. The probability of such fluctuations changes to a different degree during hemoglobin (Hb) oxygenation, changes in pH, and splitting of the intersubunit contracts. For comparison with the effect of heme removal, the rate of the hydrogen-deuterium (H-D) exchange of peptide H atoms (PHs) of human apoHb was studied by IR spectroscopy at pH 5.5–9.0 and temperatures of 10–38°C. The removal of heme increased the H-D exchange rate for 80% of Hb PHs with the exchange retardation factor P ～ 10²-10⁸. For the majority of PHs, the probability of local fluctuations depended weakly on the temperature; changes in enthalpy upon such local conformational transitions were ΔH _op ^o = 0–15 kcal/M. Global fluctuations, displaying a stronger temperature dependence, did not arise with an increase in temperature to 38°C at pH 7.0, although apoHb began slowly denaturing and aggregating under these conditions. Destabilization of the apoHb structure with a concurrent decrease in pH to 5.5 and temperature to 10°C intensified global fluctuations in the native protein structure with ΔH _op ^o < 0. The mechanism underlying the overall intensification of local fluctuations upon the heme removal, the specific features of apoHb heat denaturation under conditions close to those of in vivo Hb self-assembly, and the analogies between low-temperature global fluctuations and cold denaturation of globular proteins are discussed.     

Conformational dynamics of the tetrameric hemoglobin molecule as revealed by hydrogen exchange: 2. Effect of intersubunit contact cleavage

IR spectroscopy was used to study the rate of hydrogen-deuterium (H-D) exchange of peptide NH atoms in isolated α and β subunits of human hemoglobin (Hb) at pH 5.5–9.0 and 20°C. The H-D exchange occurs by the EX2 mechanism. The retardation factor of subunit exchange rate (P) is in a range of approximately 10²–10⁷. Compared to tetrameric Hb, the probability of local fluctuations (1/P) increases to a slightly greater extent in monomeric α subunits than in tetrameric β subunits. Unlike in the whole Hb molecule, oxygenation of its subunits has no effect on the probability of local fluctuations, and the subunits show no pH-dependent changes in 1/P values (observed for liganded Hb). Probable mechanisms accounting for overall intensification of local fluctuations upon the cleavage of contacts between subunits of the tetrameric Hb molecule are discussed with regard to structural crystallographic data.     

Equilibrium and kinetic measurements of carbon monoxide binding to hemoglobin kansas in the presence of inositol hexaphosphate

Previous proton nuclear magnetic resonance (nmr) studies have indicated that inositol hexaphosphate (IHP) can stabilize hemoglobin (Hb) Kansas in a deoxy-like quaternary structure even when fully liganded with carbon monoxide (CO) (S. Ogawa, A. Mayer, and R. G. Shulman, 1972, Biochem. Biophys. Res. Commun., 49, 1485–1491). In the present report we have investigated both CO binding at equilibrium and the CO binding and release kinetics to determine if Hb Kansas + IHP is devoid of cooperativity, as would be suggested by the nmr studies just quoted. The equilibrium measurements show that Hb Kansas + IHP has a very low affinity for CO ($\text{P}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}\text{= 1.2}\text{mm}$ Hg and K_eq = 5.4 × 10⁵M^?1) and almost no cooperativity (n = 1.1) at pH 7, 25 °C. The CO “on” and “off” kinetics also show no evidence for cooperativity. In addition, the equilibrium constant estimated from the kinetic rate constants (K_eq = 5.2 × 10⁵M^?1 with k_on = 1.03 × 10⁵M^?1 · S^? and k_off = 0.198 S^?1) is in excellent agreement with the equilibrium constant determined directly. Thus, both kinetic and equilibrium measurements allow us to conclude that CO binding to Hb Kansas + IHP occurs without significant cooperativity.     

A role of heme side-chains of human hemoglobin in its function revealed by circular dichroism and resonance Raman spectroscopy

Structural changes of heme side-chains of human adult hemoglobin (Hb A) upon ligand (O₂ or CO) dissociation have been studied by circular dichroism (CD) and resonance Raman (RR) spectroscopies. We point out the occurrence of appreciable deformation of heme side-chains like vinyl and propionate groups prior to the out-of-plane displacement of heme iron. Referring to the recent fine resolved crystal structure of Hb A, the deformations of heme side-chains take place only in the β subunits. However, these changes are not observed in the isolated β chain (β₄ homotetramer) and, therefore, are associated with the α–β inter-subunit interactions. For the communications between α and β subunits in Hb A regarding signals of ligand dissociation, possible routes are proposed on the basis of the time-resolved absorption, CD, MCD (magnetic CD), and RR spectroscopies. Our finding of the movements of heme side-chains would serve as one of the clues to solve the cooperative O₂ binding mechanism of Hb A.     

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.