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S Saigo

Publications and source records attributed to S Saigo.

18 recordsLinked to original sources
PubMed2001

Direct observation of two distinct affinity conformations in the T state human deoxyhemoglobin.

The main features of cooperative oxygenation of human hemoglobin have been described by assuming the equilibrium between two affinity conformations of the entire molecule, T and R. However, the molecular basis for explaining the wide variation in the O(2) affinities of the deoxy T state has remained obscure. We address this long-standing issue by trapping the conformational states of deoxyhemoglobin molecules within wet porous transparent silicate sol-gels. The equilibrium O(2) binding measurements of the encapsulated deoxyhemoglobin samples showed that deoxyhemoglobin free of anions coexists in two conformations that differ in O(2) affinity by 40 times or more, and addition of inositol hexaphosphate to this anion-free deoxyhemoglobin brings about a very slow redistribution of these affinity conformations. These results are the first, direct demonstration of the existence of equilibrium between two (at least two) functionally distinguishable conformational states in the T state deoxyhemoglobin.

Allosteric Regulation↗
PubMed1998

Asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): the issue of valency exchange.

A new framework for hemoglobin cooperativity was proposed by Ackers and colleagues on the basis of the hyper thermodynamic stability and deoxy (T) quaternary structure of one of diliganded deoxy-cyanomet hybrid hemoglobins, (alpha+CN-beta+CN-)(alpha beta), studied by hybridization of the equimolar mixture of deoxyhemoglobin and cyanomethemoglobin through a long (70-100 h) dimer exchange reaction [Daugherty et al. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. Recently, we reported that the published hyperstability of (alpha+CN-beta+CN-)(alpha beta) is incorrect due to the occurrence of valency exchange between the heme sites of both parental hemoglobins during the long deoxy incubation [Shibayama et al. (1997) Biochemistry 36, 4375-4381]. We also noted a difficulty in maintaining both anaerobicity and excess free cyanide of the sample during the long incubation, which led to formation of cyanide-unbound aqometheme in the original deoxyhemoglobin resulting from the electron transfer to cyanometheme. This paper is a response to a recent argument against our work [Ackers et al. (1997) Biochemistry 36, 10822-10829]. Ackers et al. have claimed that no appreciable formation of aqomethemoglobin with their methods ensures their sample integrity, based on a supposition that our observed valency exchange may have occurred via aqometheme. In this paper, however, we demonstrate that appreciable (>27%) valency exchange really occurs between deoxy and cyanometheme sites during 72 h incubation under conditions where both anaerobicity and excess free cyanide of the sample solution are maintained by a continuous flow of humidified N2 with HCN. This confirms our view that previous experimental data on (alpha+CN-beta+CN-)(alpha beta) obtained by the long incubations should be subject to reexamination while our earlier estimation of a lower limit of free energy of (alpha+CN-beta+CN-)(alpha beta) (i.e., >/= -10.1 kcal/mol) by a rapid method (35 min) is still valid. We also suggest a possibility that the T quaternary structure of (alpha+CN-beta+CN-)(alpha beta) assigned by Ackers and colleagues using the long incubations is an artifact arising from the valency exchange. These results suggest that the putative mechanistic picture for hemoglobin cooperativity inferred from studies on deoxy-cyanomet hybrids is without foundation.

Chemical Phenomena↗
PubMed1997

Reexamination of the hyper thermodynamic stability of asymmetric cyanomet valency hybrid hemoglobin, (alpha+CN-beta+CN-)(alpha beta): no preferentially populating asymmetric hybrid at equilibrium.

It has been reported that hybridization of the equimolar mixture of cyanomethemoglobin and deoxyhemoglobin through dimer exchange reaction results in establishment of an approximately binomial (1:2:1) equilibrium distribution of these parental hemoglobins and their hybrid molecule, (alpha+CN-beta+CN-)(alpha beta), within several days under anaerobic conditions at pH 7.4, 21.5 degrees C, leading to a hyper (i.e., about 170 times) thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta) relative to the stability of the other diliganded species at pH 7.4, 21.5 degrees C [Daugherty, M. A., Shea, M. A., Johnson, J. A., LiCata, V. J., Turner, G. J., & Ackers, G. K. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 1110-1114]. To examine whether the published "binomiality" for this deoxy-cyanomet hybrid system really reflects the thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta), we used a binomial (1:2:1) equilibrium distribution of the equimolar mixture of cyanomethemoglobin and fully oxygenated hemoglobin as a starting condition, and then this system was rapidly deoxygenated. We found that the relative population of the hybrid was reduced to 8.6% of the total upon deoxygenation. It was also found that the hybridization experiment under anaerobic conditions was not allowed to continue for a long time due to a valency exchange reaction between deoxy and cyanomet derivatives. For instance, a 48 h incubation resulted in the oxidation of 44% of Fe2+ to Fe3+ hemes in the original deoxyhemoglobin and the reduction of 42% of Fe3+ to Fe2+ hemes in the original cyanomethemoglobin. These results suggest that a real distribution of the deoxy-cyanomet hybrid system at equilibrium is fairly far from 1:2:1 (binomial distribution), and the thermodynamic stability of (alpha+CN-beta+CN-)(alpha beta) is less than one-tenth of the hyperstability previously reported. In addition, most of the previous results on deoxy-cyanomet valency hybrids placed under long anaerobic conditions should be subject to reexamination due to possible valency exchange reactions.

Adult↗
PubMed1995

Oxygen equilibrium and electron paramagnetic resonance studies on copper(II)-iron(II) hybrid hemoglobins at room temperature.

Copper(II)-iron(II) hybrid hemoglobins, in which hemes in either the alpha or beta subunits are substituted with copper(II) protoporphyrin IX, have been prepared. The affinities of the ferrous-subunits in both hybrids for the first binding oxygen are as low as the affinity of deoxyhemoglobin under various solution conditions, indicating the equality of behavior in copper(II) protoporphyrin IX and deoxyheme. Electron paramagnetic resonance (EPR) examinations on these hybrids at room temperature show that the interaction between copper(II) and the proximal histidine (F8) is specifically weakened in the alpha subunits within a low affinity conformation of hemoglobin. These results suggest that copper(II) protoporphyrin IX is a useful EPR probe at room temperature for investigating the deoxyheme environment in hemoglobin.

Copper↗
PubMed1995

Fixation of the quaternary structures of human adult haemoglobin by encapsulation in transparent porous silica gels.

We have used the sol-gel method to encapsulate oxy- and deoxy haemoglobins in transparent wet porous silica gels and fixed their original functional states with the retention of the reversible oxygenation properties as well as the intact spectroscopic properties. Haemoglobin originally encapsulated in aerobic gel binds oxygen non-cooperatively with very high affinity, corresponding to that for the last oxygen molecule binding to haemoglobin in solution. In contrast, haemoglobin originally encapsulated in anaerobic gel binds oxygen non-cooperatively with very low affinity, comparable to that for the first oxygen molecule binding to haemoglobin in solution. Furthermore, a detailed comparison of visible absorption spectra of deoxygenated haemoglobins originally encapsulated in aerobic and anaerobic gels indicates the retention of their original quaternary structures during the oxygenation or deoxygenation process. These results demonstrate that oxygen affinities of oxy- and deoxyhaemoglobins in solution can be satisfactorily fixed by encapsulation in wet porous silica gels, which presumably prevents the changes in the quaternary structures of haemoglobin. In addition, these results suggest a new capability of the sol-gel method to control the structural states of a variety of proteins, and further open up a new area of investigation of protein structure-function relationships.

Adult↗
PubMed1995

Oxygen equilibrium properties of nickel(II)-iron(II) hybrid hemoglobins cross-linked between 82 beta 1 and 82 beta 2 lysyl residues by bis(3,5-dibromosalicyl)fumarate: determination of the first two-step microscopic Adair constants for human hemoglobin.

We have previously reported that cross-linked asymmetric Ni(II)-Fe(II) hybrid hemoglobin, XL[alpha (Fe) beta (Fe)][alpha (Ni) beta (Ni)], in which the alpha 1 beta 1 dimer containing ferrous protoporphyrin IX and the adjacent alpha 2 beta 2 dimer containing nickel(II) protoporphyrin IX were cross-linked between Lys-82 beta 1 and Lys-82 beta 2 by reaction with bis(3,5-dibromosalicyl)fumarate, represents an adequate model for determination of the alpha 1 beta 1 oxygenation properties of native hemoglobin [Shibayama, N., Imai, K., Morimoto, H., & Saigo, S. (1993) Biochemistry 32, 8792-8798]. To extend the approach using cross-linked Ni(II)-Fe(II) hybrids to all possible pathways for initial-half oxygenation of hemoglobin, we have prepared three other types of cross-linked Ni(II)-Fe(II) hybrids, carrying nickel(II) protoporphyrin IX in two subunits and ferrous protoporphyrin IX in the other two subunits, and have determined the two-step oxygen equilibrium curves of the ferrous subunits within these cross-linked hybrids. For the first step of oxygenation, the alpha subunit shows about 3-fold higher affinity than the beta subunit at all pH values examined, indicative of a significant functional heterogeneity of the subunits in deoxyhemoglobin. For the second step of oxygenation, the cooperativity represented by the Hill coefficient (nmax) increases in the order of beta 1 beta 2 (nmax = 1.36), alpha 1 beta 1 (nmax = 1.41), alpha 1 beta 2 (nmax = 1.64), and alpha 1 alpha 2 (nmax = 1.72) at pH 7.4 in the presence of 0.1 M Cl- at 25 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspirin↗
PubMed1993

X-ray absorption spectroscopic studies of a transient intermediate in the reaction of cyanide metmyoglobin with dithionite by using rapid freezing.

The reaction of cyanide metmyoglobin (Mb+CN-) with dithionite produces a transient intermediate, supposed to be cyanide-ligated ferrous myoglobin. The Fe K-edge X-ray absorption spectrum of the intermediate has been measured by using rapid freezing and compared with those of Mb+CN- and deoxymyoglobin (deoxyMb). The shapes of the XANES (X-ray Absorption Near Edge Structure) spectra of Mb+CN- and the intermediate are very similar, including the intensity ratios of the peak C1 to D. This indicates that CN- remains bound with a linear Fe-C-N configuration in the intermediate. The absorption edge of the intermediate is shifted to 1.2 eV lower energy than that of Mb+CN-, reflecting a valence change in the heme iron. The EXAFS (Extended X-ray Absorption Fine Structure) spectrum of the intermediate closely resembles that of Mb+CN- but significantly differs from that of deoxyMb. Analysis shows that the average iron-nearest neighbor atom distance is 1.99 +/- 0.01 A for both Mb+CN- and the intermediate and 2.05 +/- 0.01 A for deoxyMb. These results imply that the local structure around the heme iron of Mb+CN- does not change upon reduction until the cyanide ligand is released.

Animals↗
PubMed1993

Oxygen equilibrium properties of asymmetric nickel(II)-iron(II) hybrid hemoglobin.

Asymmetric Ni(II)-Fe(II) hybrid hemoglobin, XL[alpha(Fe)beta(Fe)][alpha(Ni)beta(Ni)], in which the alpha 1 beta 1 dimer containing ferrous protoporphyrin IX and the complementary alpha 2 beta 2 dimer containing Ni(II) protoporphyrin IX were cross-linked between Lys-82 beta 1 and Lys-82 beta 2 by reaction with bis(3,5-dibromosalicyl) fumarate, was synthesized and characterized. We have previously shown that (i) Ni(II) protoporphyrin IX, which binds neither oxygen nor carbon monoxide, mimics a fixed deoxyheme with respect to its effect on the oxygen equilibrium properties of the counterpart iron subunits in both symmetric Ni(II)-Fe(II) hybrid Hbs [Shibayama, N., Morimoto, H., & Miyazaki, G. (1986) J. Mol. Biol. 192, 323-329] and (ii) the cross-linking used in this study little affects the oxygen equilibrium properties of hemoglobin [Shibayama, N., Imai, K., Hirata, H., Hiraiwa, H., Morimoto, H., & Saigo, S. (1991) Biochemistry 30, 8158-8165]. These remarkable features of our model allowed us to measure the oxygen equilibrium curves for the first two steps of oxygen binding to the alpha 1 beta 1 dimer within the hemoglobin tetramer. At all pH values examined, the affinities of this asymmetric hybrid for the first oxygen molecule are as low as those of native hemoglobin. The hybrid did not show cooperative oxygen binding at pH 6.4, while significant cooperativity was observed with rising pH; i.e., the Hill coefficient was increased from 1.41 to 1.53 upon a pH change from 7.4 to 8.4. The electronic absorption spectrum of Ni(II) protoporphyrin IX in the alpha 2 subunit was changed upon carbon monoxide (or oxygen) binding to the alpha 1 beta 1 dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Cross-Linking Reagents↗
PubMed1992

Effects of K-76COOH (MX-1) on immune response: induction of suppressor T-cells by MX-1.

K-76COOH (MX-1), isolated from the cultured supernatant of a species of fungi imperfecti, Stachybotrys complement nov. sp. K-76, is an inhibitor of the complement component, C5. The effects of MX-1 on various immune responses were investigated. MX-1 enhanced the response of spleen cells to PHA and LPS: MX-1 at 0.01-250 micrograms/ml for PHA and at 10-250 micrograms/ml for LPS. In contrast, it inhibited the response to Con A: MX-1 at 0.01-500 micrograms/ml for spleen cells and at 100-500 micrograms/ml for thymocytes. MX-1 and IL-1 synergistically acted to enhance the Con A response of spleen and thymus cells from which accessory cells and Ia-positive cells had been removed by passing through Sephadex G-10 columns and treating with anti-Ia monoclonal antibody plus complement. T-cells pretreated with MX-1, IL-1 and Con A for 3 days suppressed not only the response of B-cells to LPS but also the production of anti-SRBC antibodies. In addition, MX-1 was found to increase CD8+ T-cells. These results suggest that MX-1 acts on T-cells to induce suppressor T-cells.

Animals↗
PubMed1991

Oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between 82 beta 1 and 82 beta 2 lysyl residues by bis(3,5-dibromosalicyl) fumarate.

We investigated oxygen equilibrium properties of highly purified human adult hemoglobin cross-linked between lysine-82 beta 1 and lysine-82 beta 2 by a fumaryl group, which is prepared by reaction of the CO form with bis(3,5-dibromosalicyl) fumarate. The cross-linked hemoglobin preparation isolated by the previous purification method, namely, gel filtration in the presence of 1 M MgCl2 followed by ion-exchange chromatography, was found to be contaminated with about 20% of an electrophoretically silent impurity that shows remarkably high affinity for oxygen. This impurity was separated from the desired cross-linked hemoglobin by a newly developed purification method, which utilizes a difference between the authentic hemoglobin and the impurity in reactivity of the sulfhydryl groups of cysteine-93 beta toward N-ethylmaleimide under a deoxygenated condition. After this purification procedure, the oxygen equilibrium properties of purified cross-linked hemoglobin in the absence of organic phosphate became very similar to those of unmodified hemoglobin with respect to oxygen affinity, cooperativity, and the alkaline Bohr effect. The functional similarity between the cross-linked hemoglobin and unmodified hemoglobin allows us to utilize this cross-linking for preparing asymmetric hybrid hemoglobin tetramers, which are particularly useful as intermediately liganded models. Previous studies on this type of cross-linked hemoglobin should be subject to reexamination due to the considerable amount of the impurity.

Adult↗
PubMed1986

Isomerization and denaturation of homologous cytochromes c: correlation between local and gross conformational changes.

To elucidate the relationship between local and gross conformational changes, three types of conformational changes, i.e., alkaline isomerization, ligand replacement, and guanidine hydrochloride (GuHCl) denaturation, of a set of homologous and modified cytochromes c were investigated by spectroscopic methods. Cytochromes c examined include the horse, tuna, Candida, monomeric Saccharomyces, and disulfide-linked dimeric Saccharomyces proteins. Correlations were found between the apparent pK (pKa) for the isomerization and the equilibrium constants for the binding of imidazole and azide to the heme iron: the lower the pKa value, the higher the binding constant. This is explained by the fact that the isomerization and ligand replacement involve similar conformational changes localized around the sixth coordination position of the heme. A good correlation was also observed between the susceptibilities to local and gross conformational changes: the lower the pKa value for the isomerization, the lower the GuHCl concentration for the midpoint of the denaturation. Thermodynamic analysis suggests that this correlation is not due to the involvement of similar conformational changes in the two processes. Cooperative stabilization of the tertiary structure is proposed to interpret this correlation.

Animals↗
PubMed1981

Kinetic and equilibrium studies of alkaline isomerization of vertebrate cytochromes c.

Equilibria and kinetics of alkaline isomerization of seven ferricytochromes c from vertebrates were studied by pH-titration and pH-jump methods in the pH region of 7-12. In the equilibrium behavior, no significant difference was detected among the cytochromes c, whereas marked differences in the kinetic behavior were observed. According to the kinetic behavior of the isomerization, the cytochromes c examined fall into three classes: Group I (horse, sheep, dog and pigeon cytochromes c), Group II (tuna and bonito cytochromes c) and Group III (rhesus monkey cytochrome c). The kinetic results are interpreted in terms of the sequential scheme: Neutral form in equilibrium with fast Transient form in equilibrium with slow Alkaline form where the neutral and alkaline forms are the species stable at neutral and alkaline pH, respectively, and the transient form is a kinetic intermediate. From comparison of the primary sequences of the seven cytochromes c and the classification of these cytochromes c, it is concluded that the amino acid substitution Phe/Tyr at the 46-th position has a major influence on the kinetic behavior. In Group II and III cytochromes c, the ionization of Tyr-46 is suggested to bring about loosening of the heme crevice and thus facilitate the ligand replacement involved in the isomerization.

Amino Acid Sequence↗
PubMed1981

A transient spin-state change during alkaline isomerization of ferricytochrome c.

Kinetic difference spectra during the alkaline isomerization of ferricytochrome c were obtained by the pH-jump method in the range of 540 to 655 nm. The spectrum of the transient intermediate, which appears during the course of the isomerization, was reproduced from the spectra. The intermediate showed an intense absorption band at 600 nm, indicating that it is a high spin or mixed spin species. This is in contrast to the stable neutral and alkaline forms which are low spin species. The transient spin-state change during the isomerization was also observed upon rapid oxidation of ferrocytochrome c at alkaline pH.

Animals↗