Role of subunit interfaces in the allosteric mechanism of hemoglobin.
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We calculate the surface area buried in subunit interfaces of human deoxyhemoglobin and of horse methemoglobin. A larger surface area is buried in deoxy- than in methemoglobin as a result of tertiary and quaternary structure changes. In both molecules the dimer-dimer interface is closepacked. This implies that hydrophobicity stabilizes the deoxystructure, the free energy spent in keeping the subunits in a low-affinity conformation being compensated by hydrophobic free energy due to the smaller surface area accessible to solvent. (+info)
Rate of quaternary structure change in hemoglobin measured by modulated excitation.
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Using a novel technique of modulated photo-dissociation of carbon monoxide from hemoglobin, we have obtained the rates for conversion between the two quaternary states, R, and T, at 3-fold ligation. Our measurements at pH 7 and 22 degrees give rates of 780 +/- 40 sec-1 for going from R to T, and 2500 +/- 200 sec-1 from T to R. This yields an equilibrium constant of 0.31 +/- 0.04, which is in good agreement with previous estimates. The degree of agreement between this equilibrium constant and that predicted from the allosteric model provides a new, quantitative test of the allosteric description. A sequential model for the change in structure was found incompatible with the data, even if kinetic subunit inequivalence was assumed. The technique described here is quite general and can be used as long as the system under investigation can be repetitively excited in a regime in which it responds linearly to the excitation. (+info)
Site-directed mutagenesis in hemoglobin: test of functional homology of the F9 amino acid residues of hemoglobin alpha and beta chains.
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The cysteine residue at F9(93) of the human hemoglobin (Hb A) beta chain, conserved in mammalian and avian hemoglobins, is located near the functionally important alpha1-beta2 interface and C-terminal region of the beta chain and is reactive to sulfhydryl reagents. The functional roles of this residue are still unclear, although regulation of local blood flow through allosteric S-nitrosylation of this residue is proposed. To clarify the role of this residue and its functional homology to F9(88) of the alpha chain, we measured oxygen equilibrium curves, UV-region derivative spectra, Soret-band absorption spectra, the number of titratable -SH groups with p-mercuribenzoate and the rate of reaction of these groups with 4, 4'-dipyridine disulfide for three recombinant mutant Hbs with single amino acid substitutions: Ala-->Cys at 88alpha (rHb A88alphaC), Cys-->Ala at 93beta (rHb C93betaA) and Cys-->Thr at 93beta (rHb C93betaT). These Hbs showed increased oxygen affinities and impaired allosteric effects. The spectral data indicated that the R to T transition upon deoxygenation was partially restricted in these Hbs. The number of titratable -SH groups of liganded form was 3.2-3.5 for rHb A88alphaC compared with 2.2 for Hb A, whereas those for rHb C93betaA and rHb C93betaT were negligibly small. The reduction of rate of reaction with 4,4'-dipyridine disulfide upon deoxygenation in rHb A88alphaC was smaller than that in Hb A. Our experimental data have shown that the residues at 88alpha and 93beta have definite roles but they have no functional homology. Structure-function relationships in our mutant Hbs are discussed. (+info)
The effect of long-term streptozotocin-induced diabetes on contractile and relaxation responses of coronary arteries: selective attenuation of CGRP-induced relaxations.
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1. This study investigates the effect of partially metabolic controlled long-term (34 weeks) streptozotocin (STZ)-induced diabetes on relaxation and contractile responses of isolated coronary arteries to seven different vasoactive agents. 2. The average fasting and non-fasting blood glucose concentrations (mM) were significantly elevated in STZ-induced diabetic rats (P<0.0001; 10.4+/-0.4 and 16. 6+/-1.1, n=15) compared to those (4.3+/-0.03 and 4.7+/-0.18, n=11) in age-matched controls. The level of glycated haemoglobin (HbA(1)) was also significantly (P<0.0001) increased in STZ-induced diabetic rats. In STZ-induced diabetic rats, the HbA(1) levels were significantly correlated with the non-fasting blood glucose concentrations (r=0.76; P=0.003; n=13). In both groups, there was no significant correlation between the HbA(1) levels and maximal responses or sensitivities to the vasoactive agents. 3. The maximal relaxation induced by rat-alphacalcitonin gene-related peptide (rat-alphaCGRP) was significantly attenuated in the coronary arteries of STZ-induced diabetic rats (P<0.05; 40+/-7%, n=15) compared to that in age-matched controls (63+/-3%, n=11). However, there was no significant difference in the sensitivity to rat-alphaCGRP between the two groups. 4. There was no significant difference in either maximal response or sensitivity to any of the six other vasoactive agents between STZ- induced diabetic rats (n=15) and age-matched controls (n=11). 5. Our results show that partially metabolic controlled long-term (34 weeks) STZ-induced diabetes causes a selective depression of rat-alphaCGRP-induced relaxation in the intramural coronary arteries of Wistar rats. (+info)
Role of micronutrients in sport and physical activity.
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Many micronutrients play key roles in energy metabolism and, during strenuous physical activity, the rate of energy turnover in skeletal muscle may be increased up to 20-100 times the resting rate. Although an adequate vitamin and mineral status is essential for normal health, marginal deficiency states may only be apparent when the metabolic rate is high. Prolonged strenuous exercise performed on a regular basis may also result in increased losses from the body or in an increased rate of turnover, resulting in the need for an increased dietary intake. An increased food intake to meet energy requirements will increase dietary micronutrient intake, but athletes in hard training may need to pay particular attention to their intake of iron, calcium and the antioxidant vitamins. (+info)
Haematological and spermatotoxic effects of ethylene glycol monomethyl ether in copper clad laminate factories.
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OBJECTIVES: To investigate the effects of ethylene glycol monomethyl ether (EGME) on haematology and reproduction in exposed workers. METHODS: 53 Impregnation workers from two factories that make copper clad laminate with EGME as a solvent were recruited as the exposed group. Another group of 121 lamination workers with indirect exposure to EGME was recruited as the control group. Environmental monitoring of concentrations of EGME in air and biological monitoring of urinary methoxyacetic acid (MAA) concentrations were performed. Venous blood was collected for routine and biochemical analyses. Semen was collected from 14 workers exposed to EGME for sperm analysis and was compared with 13 control workers. RESULTS: Results of haematological examination showed that the haemoglobin, packed cell volume, and red blood cell count in the male workers exposed to EGME were significantly lower than in the controls. The frequency of anaemia in the exposed group (26.1%) was significantly higher than in the control group (3.2%). However, no differences were found between the female workers exposed and not exposed to EGME. After adjustment for sex, body mass index, and duration of employment, red blood cell count was significantly negatively associated with air concentrations of EGME, and haemoglobin, packed cell volume, and red blood cell count were significantly negatively associated with urinary concentrations of MAA. The pH of semen in the exposed workers was significantly lower than in the control workers, but there were no significant differences in the sperm count or sperm morphology between the exposed and control groups. CONCLUSION: It can be concluded that EGME is a haematological toxin, which leads to anaemia in the exposed workers. However, the data from this study did not support the theory of a spermatotoxic effect of EGME. (+info)
Characterization of a new electrophoretically silent hemoglobin variant. Hb saale OR alpha 2beta 2 84(EF8)Thr --> Ala.
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A new abnormal hemoglobin was detected in a young German anemic patient by cation-exchange high performance liquid chromatography (HPLC). Using a combination of electrospray mass spectrometry, HPLC, direct sequencing, and family screening with polymerase chain reaction/restriction digestion approach, we have characterized this hemoglobin variant as resulting from a Thr --> Ala replacement at beta84(EF8). It could be separated neither by electrophoresis nor by isoelectric focusing. Hb Saale is slightly unstable, exhibiting a moderate tendency to auto-oxidize. Functional properties and the heterotropic interactions are similar to those of Hb A. (+info)
Assembly of gamma- with alpha-globin chains to form human fetal hemoglobin in vitro and in vivo.
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Soluble gamma-globin chains were expressed in bacteria and purified to assess the mechanism of gamma- and alpha-chain assembly to form Hb F. Formation of Hb F in vitro following incubation of equimolar mixtures of gamma and alpha chains was about 4 x 10(5)-fold slower than assembly of alpha and beta chains to form Hb A in vitro. Results of assembly for gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains were similar to that of beta chains, whereas assembly of gamma(112Thr-->Cys) and alpha chains was similar to wild type gamma chains, indicating that amino acid differences at alpha1beta1 and alpha1gamma1 interaction sites between gamma116 Ile and beta116 His are responsible for the different assembly rates in vitro in the formation of Hb F and Hb A. Homoassembly in vitro of individual gamma chains as assessed by size-exclusion chromatography shows that gamma and gamma(112Thr-->Cys) chains form stable dimers like alphabeta and alphagamma that do not dissociate readily into monomers like beta chains. In contrast, gamma(116Ile-->His) chains form monomers and dimers upon dilution. These results are consistent with the slower assembly rate in vitro of gamma and gamma(112Thr-->Cys) with alpha chains, whereas the faster rate of assembly of gamma(116Ile-->His) and gamma(112Thr-->Asp) chains with alpha chains, like beta chains, may be caused by dissociation to monomers. These results suggest that dissociation of gamma(2) dimers to monomers limits formation of Hb F in vitro. However, yields of soluble Hb F expressed in bacteria were similar to Hb A, and no unassembled alpha and gamma chains were detected. These results indicate that gamma chains assemble in vivo with alpha chains prior to forming stable gamma(2) dimers, possibly binding to alpha chains as partially folded nascent gamma-globin chains prior to release from polyribosomes. (+info)