Dynamic Interfacial Tension Measurement for the Kinetic Study of Eu(III) Extraction in the NaNO3/Tributyl Phosphate System Based on an Increase in Interfacial Tension.

We demonstrated for the first time that interfacial tension measurements can be used to evaluate the kinetics of the solvent extraction of metal ions. The Eu(III) extraction mechanism in the nitrate ion/tributyl phosphate (TBP) system was investigated on the basis of dynamic interfacial tension. Interestingly, the interfacial tension of the TBP droplet (γ) increased with Eu(III) extraction. This behavior can be explained by the electrocapillary effect. The time dependence of γ was kinetically analyzed, and we demonstrated that the rate-determining process was the interfacial reaction of Eu(III). Furthermore, the dependence of the mass transfer rate constant upon the concentration of the nitrate ions revealed that two nitrate ions were involved in the interfacial reaction during Eu(III) extraction. However, no change in the rate constant upon TBP concentration was observed, because the change in the TBP concentration did not affect the electrocapillary effect. We determined the forward and backward reaction rates to be k1 = (1.5 ± 0.7) × 10-6 m M-2 s-1 and k-MT = (6.9 ± 3.9) × 10-7 m s-1. Therefore, for the first time, we demonstrated that dynamic interfacial tension, which is involved in the electrocapillary effect, can be used to elucidate the kinetics of Eu(III) extraction. We expect that this study will attract the attention of researchers in several fields, including physical and analytical chemistry.

Effect of Organic Solvent on the Mass Transfer Mechanism of Coumarin 102 in a Single Octadecylsilyl Silica Gel/Organic Solvent-Water System by Laser Trapping and Fluorescence Microspectroscopy.

Understanding mass transfer kinetics within individual porous particles is crucial for theoretically explaining the retention and elution behaviors in chromatography and drug delivery. Using laser trapping and fluorescence microspectroscopy, we investigated the diffusion mechanism of coumarin 102 (C102) into single octadecylsilyl particle in acetonitrile (ACN)/water, N,N-dimethylformamide (DMF)/water, and 1-butanol (BuOH)/water solutions. The intraparticle diffusion behavior of C102 was evaluated using the spherical diffusion equation, allowing us to determine the intraparticle diffusion coefficients (Dintra): (8-10) × 10-9 cm2 s-1 for ACN, (10-16) × 10-9 cm2 s-1 for DMF, and (4-6) × 10-9 cm2 s-1 for BuOH. The obtained Dintra values were further analyzed using a pore and surface diffusion model. Thus, we revealed that the diffusion mechanism of C102 differed depending on the organic solvent: surface diffusion for ACN and DMF and pore and surface diffusions for BuOH were observed. This difference is attributed to the formation of a concentrated liquid phase of ACN and DMF at the interface of the alkyl chain and the bulk solution in the pore.

Pore Size Dependence of Mass Transfer of Zinc Myoglobin in a Single Mesoporous Silica Particle.

This study investigated the pore size dependence of the mass transfer of zinc myoglobin (ZnMb) in a single mesoporous silica particle through confocal fluorescence microspectroscopy. The ZnMb's fluorescence depth profile in the particle was analyzed by a spherical diffusion model, and the intraparticle diffusion coefficient was obtained. The intraparticle diffusion coefficient in the silica particle with various pore sizes (10, 15, 30, and 50 nm) was furthermore analyzed based on a pore and surface diffusion model. Although the mass transfer mechanism in all silica particles followed the pore and surface diffusion model, the adsorption and desorption of ZnMb affected the mass transfer depending on the pore size. The influence of the slow desorption of ZnMb became pronounced for large pore sizes (30 and 50 nm), which was revealed by simulation using a diffusion equation combined with the adsorption-desorption kinetics. The distribution of ZnMb was suppressed in small pore sizes (10 and 15 nm) owing to the adsorption of ZnMb onto the entrance of the pore. Thus, we revealed the mass transfer mechanism of ZnMb in the silica particle with different pore sizes.

Heme-bound tyrosine vibrations in hemoglobin M: Resonance Raman, crystallography, and DFT calculation.

Hemoglobins M (Hbs M) are human hemoglobin variants in which either the α or ß subunit contains a ferric heme in the α2ß2 tetramer. Though the ferric subunit cannot bind O2, it regulates O2 affinity of its counterpart ferrous subunit. We have investigated resonance Raman spectra of two Hbs, M Iwate (α87His → tyrosine [Tyr]) and M Boston (α58His → Tyr), having tyrosine as a heme axial ligand at proximal and distal positions, respectively, that exhibit unassigned resonance Raman bands arising from ferric (not ferrous) hemes at 899 and 876 cm-1. Our quantum chemical calculations using density functional theory on Fe-porphyrin models with p-cresol and/or 4-methylimidazole showed that the unassigned bands correspond to the breathing-like modes of Fe3+-bound Tyr and are sensitive to the Fe-O-C(Tyr) angle. Based on the frequencies of the Raman bands, the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston were predicted to be 153.5° and 129.2°, respectively. Consistent with this prediction, x-ray crystallographic analysis showed that the Fe-O-C(Tyr) angles of Hbs M Iwate and M Boston in the T quaternary structure were 153.6° and 134.6°, respectively. It also showed a similar Fe-O bond length (1.96 and 1.97 Å) and different tilting angles.

Direct Quantification of Proteins Modified on a Polystyrene Microparticle Surface Based on ζ Potential Change.

The zeta potential (ζ) of a particle is a surface charge density (σ)-dependent parameter. If a change in σ can be induced by surface modification, the number of molecules modified on the particle can be detected as a measurable change in ζ. In this study, we demonstrate protein detection at zmol or pM levels (bovine serum albumin (BSA), myoglobin (Mb), and lysozyme (Lyz)) on carboxy-functionalized polystyrene (PS) microparticles using the ζ change. Protein modification of the PS particles changes σ because the negatively charged carboxy group is used for protein binding, and proteins also have charged amino acids. The pH dependence of ζ for the protein-modified particles at 4 < pH < 10 is well-explained using the acid dissociation of the acidic and basic amino acids and the Gouy-Chapman-Stern model. An increase in the binding number of proteins per single PS particle (npro/PS) leads to a decrease in ζ, which is consistent with the results estimated by the proposed model. The detection limits of nBSA/PS, nMb/PS, and nLyz/PS are 1.17 × 104, 1.22 × 104, and 1.20 × 104 at pH 8.52, respectively, which means that the concentration-based detection limits are 722, 376, and 371 pM, respectively. We expect that the present method will be a strategy for the detection of molecules on particles.

Distribution Behavior of Single-Stranded DNA Molecules in an Amino-Group-Functionalized Silica Microparticle.

In this study, we investigated the distribution behavior of single-stranded DNA molecules with 20 bases in silica particles (particle size: ∼30 µm) using confocal fluorescence microspectroscopy. The distribution kinetics was investigated under various conditions, such as the type of base (adenine, thymine, guanine, and cytosine), pore size of the particle (30 and 50 nm), and salt concentration (100, 200, and 500 mM), which changed the distribution behavior. At high salt concentrations, we observed sigmoidal kinetic behavior, which does not occur in the general distribution of small organic molecules but is often observed in protein aggregation and nuclear growth. An analytical model based on DNA aggregation explained the sigmoidal distribution behavior well, and this model also worked well when the number of DNA molecules involved in DNA aggregation was greater than two. The intraparticle diffusion of DNA molecules was analyzed using the pore and surface diffusion model. As a result, the intraparticle diffusion of DNA aggregates mainly occurs according to surface diffusion, and the surface diffusion coefficient has the same value ((2.4-6.7) × 10-9 cm2 s-1) independent of the pore size and type of base.

Semi-quantification of the binding constant based on bond breaking in a combined acoustic-gravitational field.

In this study, we propose a semi-quantification method based on breaking bonds between microparticles and glass plates in a combined acoustic-gravitational field. The semi-quantified binding constant values for BSA-ibuprofen, BSA-ciprofloxacin, ConA-glycogen, ConA-mannan, and BSA-naproxen calculated using this method were 7.5 × 103, 1.6 × 104, 2.3 × 105, 2.4 × 106 and 9.0 × 107 M-1, respectively, which were in concord with the reported values.

Roles of Fe-Histidine bonds in stability of hemoglobin: Recognition of protein flexibility by Q Sepharose.

Using various mutants, we investigated to date the roles of the Fe-histidine (F8) bonds in cooperative O2 binding of human hemoglobin (Hb) and differences in roles between α- and ß-subunits in the α2ß2 tetramer. An Hb variant with a mutation in the heme cavity exhibited an unexpected feature. When the ß mutant rHb (ßH92G), in which the proximal histidine (His F8) of the ß-subunit is replaced by glycine (Gly), was subjected to ion-exchange chromatography (Q Sepharose column) and eluted with an NaCl concentration gradient in the presence of imidazole, yielded two large peaks, whereas the corresponding α-mutant, rHb (αH87G), gave a single peak similar to Hb A. The ß-mutant rHb proteins under each peak had identical isoelectric points according to isoelectric focusing electrophoresis. Proteins under each peak were further characterized by Sephadex G-75 gel filtration, far-UV CD, 1H NMR, and resonance Raman spectroscopy. We found that rHb (ßH92G) exists as a mixture of αß-dimers and α2ß2 tetramers, and that hemes are released from ß-subunits in a fraction of the dimers. An approximate amount of released hemes were estimated to be as large as 30% with Raman relative intensities. It is stressed that Q Sepharose columns can distinguish differences in structural flexibility of proteins having identical isoelectric points by altering the exit rates from the porous beads. Thus, the role of Fe-His (F8) bonds in stabilizing the Hb tetramer first described by Barrick et al. was confirmed in this study. In addition, it was found in this study that a specific Fe-His bond in the ß-subunit minimizes globin structural flexibility.

Kinetic Analysis of the Mass Transfer of Zinc Myoglobin in a Single Mesoporous Silica Particle by Confocal Fluorescence Microspectroscopy.

The adsorption/desorption mechanisms of biomolecules in porous materials have attracted significant attention because of their applications in many fields, including environmental, medical, and industrial sciences. Here, we employ confocal fluorescence microspectroscopy to reveal the diffusion behavior of zinc myoglobin (ZnMb, 4.4 nm × 4.4 nm × 2.5 nm) as a spherical protein in a single mesoporous silica particle (pore size of 15 nm). The measurement of the time course of the fluorescence depth profile of the particle reveals that intraparticle diffusion is the rate-limiting process of ZnMb in the particle. The diffusion coefficients of ZnMb in the particle for the distribution (Ddis) and release (Dre) processes are determined from the rate constants, e.g., Ddis = 1.65 × 10-10 cm2 s-1 and Dre = 3.68 × 10-10 cm2 s-1, for a 10 mM buffer solution. The obtained D values for various buffer concentrations are analyzed using the pore and surface diffusion model. Although surface diffusion is the main distribution process, the release process involves pore and surface diffusion, which have not been observed with small organic molecules; the mechanism of transfer of small molecules is pore diffusion alone. We demonstrate that the mass transfer kinetics of ZnMb in the silica particle can be explained well on the basis of pore and surface diffusion.

Interrelationship among Fe-His Bond Strengths, Oxygen Affinities, and Intersubunit Hydrogen Bonding Changes upon Ligand Binding in the ß Subunit of Human Hemoglobin: The Alkaline Bohr Effect.

Regulation of the oxygen affinity of human adult hemoglobin (Hb A) at high pH, known as the alkaline Bohr effect, is essential for its physiological function. In this study, structural mechanisms of the alkaline Bohr effect and pH-dependent O2 affinity changes were investigated via 1H nuclear magnetic resonance and visible and UV resonance Raman spectra of mutant Hbs, Hb M Iwate (αH87Y) and Hb M Boston (αH58Y). It was found that even though the binding of O2 to the α subunits is forbidden in the mutant Hbs, the O2 affinity was higher at alkaline pH than at neutral pH, and concomitantly, the Fe-His stretching frequency of the ß subunits was shifted to higher values. Thus, it was confirmed for the ß subunits that the stronger the Fe-His bond, the higher the O2 affinity. It was found in this study that the quaternary structure of α(Fe3+)ß(Fe2+-CO) of the mutant Hb is closer to T than to the ordinary R at neutral pH. The retained Aspß94-Hisß146 hydrogen bond makes the extent of proton release smaller upon ligand binding from Hisß146, known as one of residues contributing to the alkaline Bohr effect. For these T structures, the Aspα94-Trpß37 hydrogen bond in the hinge region and the Tyrα42-Aspß99 hydrogen bond in the switch region of the α1-ß2 interface are maintained but elongated at alkaline pH. Thus, a decrease in tension in the Fe-His bond of the ß subunits at alkaline pH causes a substantial increase in the change in global structure upon binding of CO to the ß subunit.

Heterogeneity between Two α Subunits of α2ß2 Human Hemoglobin and O2 Binding Properties: Raman, 1H Nuclear Magnetic Resonance, and Terahertz Spectra.

Following a previous detailed investigation of the ß subunit of α2ß2 human adult hemoglobin (Hb A), this study focuses on the α subunit by using three natural valency hybrid α(Fe2+-deoxy/O2)ß(Fe3+) hemoglobin M (Hb M) in which O2 cannot bind to the ß subunit: Hb M Hyde Park (ß92His → Tyr), Hb M Saskatoon (ß63His → Tyr), and Hb M Milwaukee (ß67Val → Glu). In contrast with the ß subunit that exhibited a clear correlation between O2 affinity and Fe2+-His stretching frequencies, the Fe2+-His stretching mode of the α subunit gave two Raman bands only in the T quaternary structure. This means the presence of two tertiary structures in α subunits of the α2ß2 tetramer with T structure, and the two structures seemed to be nondynamical as judged from terahertz absorption spectra in the 5-30 cm-1 region of Hb M Milwaukee, α(Fe2+-deoxy)ß(Fe3+). This kind of heterogeneity of α subunits was noticed in the reported spectra of a metal hybrid Hb A like α(Fe2+-deoxy)ß(Co2+) and, therefore, seems to be universal among α subunits of Hb A. Unexpectedly, the two Fe-His frequencies were hardly changed with a large alteration of O2 affinity by pH change, suggesting no correlation of frequency with O2 affinity for the α subunit. Instead, a new Fe2+-His band corresponding to the R quaternary structure appeared at a higher frequency and was intensified as the O2 affinity increased. The high-frequency counterpart was also observed for a partially O2-bound form, α(Fe2+-deoxy)α(Fe2+-O2)ß(Fe3+)ß(Fe3+), of the present Hb M, consistent with our previous finding that binding of O2 to one α subunit of T structure α2ß2 tetramer changes the other α subunit to the R structure.

Utility of heme analogues to intentionally modify heme-globin interactions in myoglobin.

Myoglobin reconstitution with various synthetic heme analogues was reviewed to follow the consequences of modified heme-globin interactions. Utility of dimethyl sulfoxide as the solvent for water-insoluble hemes was emphasized. Proton NMR spectroscopy revealed that loose heme-globin contacts in the heme pocket eventually caused the dynamic heme rotation around the iron-histidine bond. The full rotational rate was estimated to be about 1400 s(-1) at room temperature for 1,4,5,8-tetramethylhemin. The X-ray analysis of the myoglobin containing iron porphine, the smallest heme without any side chains, showed that the original globin fold was well conserved despite the serious disruption of native heme-globin contacts. Comparison between the two myoglobins with static and rotatory prosthetic groups indicated that the oxygen and carbon monoxide binding profiles were almost unaffected by the heme motion. On the other hand, altered tetrapyrrole array of porphyrin dramatically changed the dissociation constant of oxygen from 0.0005 mm Hg of porphycene-myoglobin to ∞ in oxypyriporphyrin-myoglobin. Heme-globin interactions in myoglobin were also monitored with circular dichroism spectroscopy. The observation on several reconstituted protein revealed an unrecognized role of the propionate groups in protoheme. Shortening of heme 6,7-propionates to carboxylates resulted in almost complete disappearance of the positive circular dichroism band in the Soret region. The theoretical analysis suggested that the disappeared circular dichroism band reflected the cancellation effects between different conformers of the carboxyl groups directly attached to heme periphery. The above techniques were proposed to be applicable to other hemoproteins to create new biocatalysts. This article is part of a Special Issue entitled Biodesign for Bioenergetics--the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Photoinduced Bilayer-to-Nonbilayer Phase Transition of POPE by Photoisomerization of Added Stilbene Molecules.

The photocontrol of a bilayer-to-nonbilayer phase transition (the liquid-crystalline Lα phase to the inverted hexagonal HII phase) of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE) by the photoisomerization of incorporated stilbene molecules was examined by utilizing differential scanning calorimetry, small-angle X-ray diffraction, ultraviolet (UV)/visible absorption, and attenuated total reflectance Fourier transform infrared spectroscopies. cis-Stilbene lowered the transition temperature, Th, to a greater extent than did trans-stilbene, and the difference was at most ca. 10 °C. At temperatures higher than the Th of POPE/cis-stilbene but lower than that of POPE/trans-stilbene, the photoisomerization from the trans to the cis form of the stilbene molecules by irradiation with UV light caused a Lα-HII phase transition. The UV irradiation partially induced the HII phase at a constant temperature because of the incomplete photoisomerization of stilbene (ca. 60%). The reduction in Th by the incorporation of stilbenes was caused mainly by the reduction in the spontaneous radius of curvature of the lipid monolayer, R0. The greater bulkiness of cis-stilbene as compared to the trans form resulted in a more effective reduction in R0 and stabilization of the HII phase.

Heme Orientation of Cavity Mutant Hemoglobins (His F8 → Gly) in Either α or ß Subunits: Circular Dichroism, (1) H NMR, and Resonance Raman Studies.

Native human adult hemoglobin (Hb A) has mostly normal orientation of heme, whereas recombinant Hb A (rHb A) expressed in E. coli contains both normal and reversed orientations of heme. Hb A with the normal heme exhibits positive circular dichroism (CD) bands at both the Soret and 260-nm regions, while rHb A with the reversed heme shows a negative Soret and decreased 260-nm CD bands. In order to examine involvement of the proximal histidine (His F8) of either α or ß subunits in determining the heme orientation, we prepared two cavity mutant Hbs, rHb(αH87G) and rHb(ßH92G), with substitution of glycine for His F8 in the presence of imidazole. CD spectra of both cavity mutant Hbs did not show a negative Soret band, but instead exhibited positive bands with strong intensity at the both Soret and 260-nm regions, suggesting that the reversed heme scarcely exists in the cavity mutant Hbs. We confirmed by (1) H NMR and resonance Raman (RR) spectroscopies that the cavity mutant Hbs have mainly the normal heme orientation in both the mutated and native subunits. These results indicate that the heme Fe-His F8 linkage in both α and ß subunits influences the heme orientation, and that the heme orientation of one type of subunit is related to the heme orientation of the complementary subunits to be the same. The present study showed that CD and RR spectroscopies also provided powerful tools for the examination of the heme rotational disorder of Hb A, in addition to the usual (1) H NMR technique. Chirality 28:585-592, 2016. © 2016 Wiley Periodicals, Inc.

Diffusion behavior of rhodamine 6G in single octadecylsilyl-functionalized silica particle revealed by fluorescence correlation spectroscopy.

We investigated the diffusion behavior of rhodamine 6G (Rh6G) within single octadecylsilyl-functionalized (ODS) silica particle in an acetonitrile (ACN)/water system using fluorescence correlation spectroscopy (FCS). FCS measurements were conducted at the center of the particle to exclusively determine the intraparticle diffusion coefficient (D). The obtained D values were analyzed based on a pore and surface diffusion model, the results of which indicate that surface diffusion primarily governs the intraparticle diffusion of Rh6G. Furthermore, an increase in the concentration of ACN (CACN) resulted in a corresponding increase in the surface diffusion coefficient (Ds), whereas the addition of NaCl did not significantly affect the Ds values. We attributed this dependence of Ds to the dielectric constant change in the interfacial liquid phase formed on the ODS layer. Specially, Ds values of (4.0 ± 0.5) × 10-7, (7.7 ± 1.1) × 10-7, (1.0 ± 0.3) × 10-6, and (1.1 ± 0.2) × 10-6 cm2 s-1 were obtained for CACN = 20, 30, 40, and 50 vol%, respectively. We anticipate that this approach will contribute to advancing research on intraparticle mass transfer mechanisms.

Evolution of myoglobin diffusion mechanisms: exploring pore and surface diffusion in a single silica particle.

This study elucidates the mass transfer mechanism of myoglobin (Mb) within a single silica particle with a 50 nm pore size at various pH levels (6.0, 6.5, 6.8, and 7.0). Investigation of Mb distribution ratio (R) and distribution kinetics was conducted using absorption microspectroscopy. The highest R was observed at pH 6.8, near the isoelectric point of Mb, as the electrostatic repulsion between Mb molecules on the silica surface decreased. The time-course absorbance of Mb in the silica particle was rigorously analyzed based on a first-order reaction, yielding the intraparticle diffusion coefficient of Mb (Dp). Dp-(1 + R)-1 plots at different pH values were evaluated using the pore and surface diffusion model. Consequently, we found that at pH 6.0, Mb diffused in the silica particle exclusively through surface diffusion, whereas pore diffusion made a more substantial contribution at higher pH. Furthermore, we demonstrated that Mb diffusion was hindered by slow desorption, associated with the electrostatic charge of Mb. This comprehensive analysis provides insights into the diffusion mechanisms of Mb at acidic, neutral, and basic pH conditions.

Kinetic analysis of mass transfer of Eu(III) in extractant-impregnated polymer-layered silica particle in multiple-ion distribution system.

The Eu(III) distribution mechanism in single extractant-impregnated polymer-layered silica particle in a complex solution containing multiple lanthanide ions was investigated using fluorescence microspectroscopy, which was compared with the single-ion distribution system. The rate-determining step of the Eu(III) distribution was the reaction of Eu(III) with the two extractant molecules in the particle. The distribution mechanism and rate constants obtained in the multiple lanthanide ions-distribution system agreed with those of the single-ion distribution system.

Eu(III) transfer in single N,N,N',N'-tetraoctyldiglycolamide-impregnated polymer-coated silica particle using fluorescence microspectroscopy: transfer mechanism and effect of polymer crosslinking degree.

A microcapillary manipulation system combined with fluorescence microspectroscopy enabled us to analyze mass transfer in a single particle. In this study, we revealed the Eu(III) distribution in a single diglycolamide-derivative extractant (TODGA)-impregnated polymer-coated silica particle. The reaction of Eu(III) with two TODGA molecules in the polymer layer was the rate-limiting process, which was revealed by the relationship between the rate constants (k1 and k-1) and concentrations of Eu(III) and HNO3. The decrease in the crosslinking degree of the polymer layer caused an increase in only k-1. This indicates that hydrophilic environments at lower crosslinking degrees enhance the stability of the charged Eu(III) species such as Eu3+, Eu(NO3)2+, and Eu(NO3)2+.

Near-UV circular dichroism and UV resonance Raman spectra of individual tryptophan residues in human hemoglobin and their changes upon the quaternary structure transition.

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αß dimer: at α14, ß15, and ß37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (ß15Trp→Leu), and rHb (ß37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and ß15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas ß37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of ß37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.

Structural origin of cooperativity in human hemoglobin: a view from different roles of α and ß subunits in the α2ß2 tetramer.

This mini-review, mainly based on our resonance Raman studies on the structural origin of cooperative O2 binding in human adult hemoglobin (HbA), aims to answering why HbA is a tetramer consisting of two α and two ß subunits. Here, we focus on the Fe-His bond, the sole coordination bond connecting heme to a globin. The Fe-His stretching frequencies reflect the O2 affinity and also the magnitude of strain imposed through globin by inter-subunit interactions, which is the origin of cooperativity. Cooperativity was first explained by Monod, Wyman, and Changeux, referred to as the MWC theory, but later explained by the two tertiary states (TTS) theory. Here, we related the higher-order structures of globin observed mainly by vibrational spectroscopy to the MWC theory. It became clear from the recent spectroscopic studies, X-ray crystallographic analysis, and mutagenesis experiments that the Fe-His bonds exhibit different roles between the α and ß subunits. The absence of the Fe-His bond in the α subunit in some mutant and artificial Hbs inhibits T to R quaternary structural change upon O2 binding. However, its absence from the ß subunit in mutant and artificial Hbs simply enhances the O2 affinity of the α subunit. Accordingly, the inter-subunit interactions between α and ß subunits are nonsymmetric but substantial for HbA to perform cooperative O2 binding.