Kinetics of oxygen and carbon monoxide binding to synthetic analogs of the myoglobin and hemoglobin active sites.
(9/461)
Kinetics of reversible oxygenation and carbon monoxide complex formation of the simple heme compounds pyrroheme-N-[3-(1-imidazolyl)propyl]amide and pyrroheme-3-(3-pyridyl)propyl ester have been measured in different solvent environments. The oxygen on and off rates and equilibria of these compounds can be made to closely match those of myoglobin, of hemoglobin alpha chains, or of the various steps for hemoglobin by varying solvent environment or the basicity of the proximal base. These results suggest that the protein could alter oxygen on rates by varying the basicity of the proximal base and the off rates by changing the polarity of the distal environment. (+info)
Structure-function relations in hemoglobin as determined by x-ray absorption spectroscopy.
(10/461)
Conclusions concerning the structure around the iron atom in oxy- and carbonmonoxyhemoglobin have been obtained by fluorescent x-ray absorption studies. The bis-imidazole heme complex was used as a model system of known structure. The ligated forms of hemoglobin, and cytochrome c at high pH, gave spectra which were very similar to the bis-imidazole complex, where the average Fe-N bond distance is known to be 1.98 A. By comparison it was possible to determine that the average Fe-N bond distances were 1.99 A in oxyhemoglobin, 1.98 A in carbonmonoxyhemoglobin, and 1.98 A in cytochrome c at pH less than 10.5, with an experimental accuracy of +/-0.02 A. An experimental comparison between oxy- and deoxyhemoglobin A showed much larger spectral changes than amongst the ligated forms. A comparison was made between the low oxygen affinity form of deoxy HbA and the high affinity form of doexy Hb Kempsey (alpha2beta992 Asp leads to Asn). All the spectral features coincided, allowing us to conclude that the average iron-ligand bond differences must be less than or equal to 0.02 A. Since the strain energy is proportional to the square of this displacement, we show that the strain energy at the iron is less than or equal to 4 X 10(-3) eV. This is negligible compared to the difference of binding energy of the high and low affinity forms, which is 0.15 eV, showing that the energies responsible for the increase of oxygen affinity are not localized at the heme. (+info)
Mass carbon monoxide poisoning.
(11/461)
The largest occurrence of carbon monoxide poisoning in Britain demonstrates the potential for mass accidental poisoning. It emphasises the need for strict public health controls and the importance of good liaison between emergency services to ensure that such events are quickly recognised and that the necessary resources are organised. (+info)
The measurement of exhaled carbon monoxide is influenced by airflow obstruction.
(12/461)
The concentration of carboxyhaemoglobin (COHb) is often estimated from measurements of carbon monoxide in the exhaled air (COexh). This study investigates whether the presence of airflow obstruction significantly alters the relationship between COexh and COHb. Eighty-one regular smokers were prospectively studied and divided in four groups according to the presence and severity of airflow obstruction (none, mild, moderate, severe). In each subject, the authors measured in this order: 1) arterial blood gases; 2) haemoglobin concentration and COHb (by co-oxymetry); 3) COexh; 4) lung volumes; and 5) forced spirometry. The size of the measurement error (deltaCO) was calculated from the difference between COHb and COexh. Neither the smoking history nor COexh were different in the four groups of subjects studied. In contrast, deltaCO increased in parallel to the degree of airflow obstruction. DeltaCO was >2% (a threshold value normally used in the clinic to separate smokers from nonsmokers) only in patients with severe airflow obstruction. A stepwise multivariate analysis showed that both forced expiratory volume in one second (FEV1) (percentage reference) and COHb contributed significantly (p<0.0001) to predict deltaCO. This study shows that the estimation of carboxyhaemoglobin from exhaled carbon monoxide measurements can be inaccurate in patients with severe airflow obstruction. In these patients, the direct measurement of carboxyhaemoglobin seems advisable in clinical practice. (+info)
Glycosylation of hemoglobin in vitro: affinity labeling of hemoglobin by glucose-6-phosphate.
(13/461)
To determine the mechanism for the formation of hemoglobin A1c (Hb A1c) in vivo, we incubated human hemoglobin with glucose and metabolites of glucose. [14C]Glucose-6-phosphate (G6P) reacted readily with deoxyhemoglobin, and formed a covalent linkage. The reaction rate was considerably reduced in the presence of carbon monoxide or 2,3-diphosphoglycerate (2,3-DPG). Purified G6P hemoglobin had a lowered oxygen affinity and decreased reactivity with 2,3-DPG compared to Hb A. G6P behaved as a 2,3-DPG analog and reacted specifically at the NH2-terminal amino group of the beta chain. In contrast, the interaction of hemoglobin with glucose was much slower, and was unaffected by carbon monoxide or 2,3-DPG. Neither glucose-1-phosphate, fructose-6-phosphate, nor fructose-1,6-diphosphate formed a reaction product with hemoglobin. G6P behaves as an affinity label with the phosphate group forming electrostatic bonds at the 2,3-DPG binding site and the aldehvde group reacting with the NH2-terminal amino group of the beta chain. Thus, G6P hemoglobin may be an intermediate in the conversion of Hb A to Hb A1c. (+info)
Rate of quaternary structure change in hemoglobin measured by modulated excitation.
(14/461)
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.
(15/461)
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)
Multiple geminate ligand recombinations in human hemoglobin.
(16/461)
The geminate ligand recombination reactions of photolyzed carbonmonoxyhemoglobin were studied in a nanosecond double-excitation-pulse time-resolved absorption experiment. The second laser pulse, delayed by intervals as long as 400 ns after the first, provided a measure of the geminate kinetics by rephotolyzing ligands that have recombined during the delay time. The peak-to-trough magnitude of the Soret band photolysis difference spectrum measured as a function of the delay between excitation pulses showed that the room temperature kinetics of geminate recombination in adult human hemoglobin are best described by two exponential processes, with lifetimes of 36 and 162 ns. The relative amounts of bimolecular recombination to T- and R-state hemoglobins and the temperature dependence of the submicrosecond kinetics between 283 and 323 K are also consistent with biexponential kinetics for geminate recombination. These results are discussed in terms of two models: geminate recombination kinetics modulated by concurrent protein relaxation and heterogeneous kinetics arising from alpha and beta chain differences. (+info)