Biomedical subjects
V S Sharma
Publications and source records attributed to V S Sharma.
Studies on cytosolic guanylate cyclase from human placenta.
We have purified the soluble form of guanylate cyclase from human placenta greater than 2400-fold. The enzyme shared several characteristics with the enzyme purified from other sources including molecular mass and subunit composition, activation by divalent cations, inhibition by ATP and Michaelis constants. The enzyme, however, had a lower absorption maximum in the Soret region (417 +/- 1 nm) than the enzyme from other sources and was activated only one-fifth as much by nitric oxide as the bovine lung enzyme. It appears that the heme prosthetic group in the human placental enzyme may be hexa-coordinate and in the bovine lung enzyme the heme group may be penta-coordinate.
Quaternary structure and geminate recombination in hemoglobin: flow-flash studies on alpha 2CO beta 2 and alpha 2 beta 2CO.
The kinetics of geminate recombination for the diliganded species alpha 2CO beta 2 and alpha 2 beta 2CO of human hemoglobin were studied using flash photolysis. The unstable diliganded species were generated just before photolysis by chemical reduction in a continuous flow reactor from the more stable valency hybrids alpha 2CO beta 2+ and alpha 2+ beta 2CO, which could be prepared by high pressure liquid chromatography. Before the flash photolysis studies, the hybrids had been characterized by double-mixing stopped-flow kinetics experiments. At pH 6.0 in the presence of inositol hexaphosphate (IHP) both of the diliganded species show second order kinetics for overall addition of a third CO that is clearly characteristic of the T state (l' = 1-2 x 10(5) M-1 s-1), whereas at higher pH and in the absence of IHP they show combination rates characteristic of an R state. The kinetics of geminate recombination following photolysis of a bound CO, however, showed little dependence on pH and IHP concentration. This surprising observation is explained on the basis that the kinetics of geminate recombination of CO primarily depends on the tertiary structure of the ligand binding site, which apparently does not differ much between the R state and the liganded T state formed on adding IHP in this system. Since this explanation requires distinguishing different tertiary structures within a particular quaternary structure, it amounts to a contradiction to the two-state allosteric model.
A new mode for heme-heme interactions in hemoglobin associated with distal perturbations.
The distal side of the heme pocket, known to regulate ligand affinity, is shown to be directly involved in subunit interactions. Valency hybrids with oxygen or carbon monoxide bound to the reduced chain are used to model R-state hemoglobin with different distal perturbations. Electron paramagnetic resonance of the oxidized chains shows that the carbon monoxide perturbation is transmitted between subunits to the distal histidine and the oxidized iron center. A comparison of hybrids with only one type of chain oxidized and hybrids with a single alpha beta dimer oxidized is consistent with this perturbation being transmitted across the alpha 1 beta 1 interface. This represents a new mode of subunit interactions in hemoglobin.
Gastrointestinal hemorrhage in AIDS: arteriographic diagnosis and transcatheter treatment.
The usefulness of arteriography and transcatheter treatment was studied in nine patients with gastrointestinal hemorrhage and acquired immunodeficiency syndrome (AIDS). Selective arteriography was performed in all patients; transcatheter treatment was performed by means of embolotherapy or selective vasopressin infusion. Medical records were reviewed to determine the cause of hemorrhage and clinical outcome. Arteriography enabled identification of the site of hemorrhage in seven patients. Hemorrhage was caused by Kaposi sarcoma (n = 2), cytomegalovirus colitis (n = 1), lymphoma (n = 2), or unknown causes (n = 4). Neovascularity and dense parenchymal stain were present in patients with Kaposi sarcoma. Transcatheter treatment consisted of embolization (n = 3), vasopressin infusion (n = 2), or both (n = 1). Hemorrhage was controlled in six cases in which transcatheter treatment was administered. Complications included thrombosis of the femoral artery in a 7-month-old infant and formation of a pseudoaneurysm of the femoral artery, which was treated successfully with ultrasound-guided compression. In patients with AIDS and profuse gastrointestinal hemorrhage, arteriography often enables identification of a specific site of hemorrhage, which can be stopped with transcatheter treatment.
Double-mixing kinetic studies of the reactions of monoliganded species of hemoglobin: alpha 2(CO)1 beta 2 and alpha 2 beta 2(CO)1.
The kinetics of CO association to and dissociation from the two isomers of monoliganded species alpha ICO beta I(alpha II beta II) and alpha I beta I (alpha II beta COII) has been studied by double-mixing stopped-flow and microperoxidase methods. The monoliganded species were generated by hybridization between excess ferric Hb and alpha CO2 beta +2 or alpha +2 beta CO2 prepared by high-pressure liquid chromatography (HPLC). The results indicated that: 1) there were no significant differences in the reactivities of alpha and beta chains in the first step of ligation; 2) in the second step of ligation there was significant cooperativity in the reaction of deoxyhemoglobin with 0.05 or 0.1 equivalent of CO. Diliganded species were therefore formed in significant amounts. The double-mixing HPLC results suggested that in the second step of ligation alpha chains reacted faster than the beta chains, and the main diliganded species formed was alpha I beta ICO (alpha IICO beta II) or its isomer alpha ICO I(alpha II beta IICO). These results seem to indicate that the reaction of the first CO is mostly random and in the second step of ligation CO binds more to the tetramers in which one beta chain is already ligated: alpha I beta I (alpha II beta II) + CO----alpha ICO beta I (alpha II beta II) and alpha I beta ICO (alpha II beta II) + CO----alpha I beta ICO (alpha IICO beta II).
Double mixing stopped-flow method for the study of equilibria and kinetics of dimer-tetramer association of hemoglobins: studies on Hb Carp, Hb A, and Hb Rothschild.
A double mixing stopped-flow method is described for studying the dimer-tetramer equilibria of oxyhemoglobins and the kinetics of association of unliganded dimers. The three hemoglobins studied were: Hb Carp, Hb A, and Hb Rothschild (Trp beta 37 (C3)----Arg). The new method reproduces the data obtained for oxyHb A by other established methods. In agreement with previous studies, the new method indicates little, if any, dissociation of oxyHb carp into dimers even in 2 M urea solutions (0.1 M Bis-Tris pH 7.0). OxyHb Rothschild, on the other hand, is extensively dissociated into dimers (K(Hb4L4 in equilibrium with 2Hb2) = 37.3 x 10(-6) M) and the rate constant for the association of deoxy dimers of Hb Rothschild is about one-tenth of the value for Hb A indicating that the deoxy tetramer of Hb Rothschild is at least 10 times more dissociated into dimers than deoxyHb A.
Double-mixing kinetic studies of the reactions of methyl isocyanide and CO with diliganded intermediates of hemoglobin: alpha 2CO beta 2 and alpha 2 beta 2CO.
Kinetics of the reactions of CO and methyl isocyanide with two diliganded intermediates of hemoglobin, alpha 2CO beta 2 and alpha 2 beta 2CO, have been studied by double-mixing and microperoxidase methods. The valency hybrids were prepared by high-pressure liquid chromatography. The reaction time courses of ligand combination and dissociation with both of the ligands were biphasic, and in CO combination reaction the zero-time amplitudes of the two phases were independent of the protein concentration. In the presence of 2 M urea the reaction time course was clearly dependent on protein concentration, as the zero-time amplitude of the fast phase increased at lower protein concentrations. These two observations indicate that little dissociation of tetramers into dimers occurs in the absence of urea. Consistent with this, the kinetic data for the reactions of CO best fit a reaction model consisting of two tetrameric species not in rapid equilibrium with each other. Various considerations, however, suggest that the reaction model is more appropriately described as 2D in equilibrium R in equilibrium T. The reaction of triliganded species (Hb4(CO)2Me1) with methyl isocyanide was monophasic, and the reaction model suggested a fast T in equilibrium R structural change after the binding of the third ligand. Although the precise structural nature of the two species remains undefined, it is concluded that the biphasicity in the reactions of the two hybrids is characteristic of the diliganded species only and is independent of the nature of the ligand.
The dissociation of NO from nitrosylhemoglobin.
The reaction between nitrosylhemoglobin and an excess of deoxymyoglobin has been used to study the kinetics of ligand dissociation from Hb4(NO)4 and Hb4(no)1 species. The kinetics of the dissociation of the first NO molecule from Hb4(no)4 was studied by the ligand replacement method. The results indicate that: (a) the ligand dissociation reaction in Hb4(NO)4 is a cooperative process. This is consistent with the results of Moore and Gibson (Moore, E.G., and Gibson, Q.H. (1977) J. Biol. Chem. 251, 2788-2794). (b) alpha and beta chains in the T state formed by adding IHP to Hb4(NO)4 show kinetic heterogeneity. (c) A similar kinetic heterogeneity is shown by alpha and beta chains in the species Hb4NO in the absence of IHP.(d) The value for the NO dissociation rate constant calculated from the slow phases observed in (b) and (c) is similar to that estimated for the R state. These results suggest that the R to T transition brought about with or without inositol hexaphosphate changes the ligand affinity of one type of the chains much more than of the other. On the basis of IR and EPR studies, it is suggested that alpha chains undergo larger functional changes in R to T transition (or vice versa) in nitrosylhemoglobin. The kinetic parameters for HbNO are compared with those of HbO2 and HbCO and the implications of the results for the reaction mechanism are discussed.
Morbidity pattern among children below 5 years in an urban Sindhi community.
Explore the source record for details and available documents.
"Tension" on heme by the proximal base and ligand reactivity: conclusions drawn from model compounds for the reaction of hemoglobin.
The kinetic data on model compounds of hemoglobin indicate that in oxyderivatives ligand dissociation rates are sensitive to the "tension" exerted by the proximal base on the metal-to-ligand bond; the corresponding rates for carboxy derivatives are not sensitive to the tension. It is suggested that the metal-to-ligand bond becomes weaker with increased "pull" (or tension) on Fe from the proximal base due to the steric and/or electronic interaction between the ligand, the porphyrin ring, and the proximal base. In model compounds the linear heme Fe-to-CO bound vis-a-vis the bent heme Fe-to-O2 bond probably makes such interactions less significant in carboxy derivatives. It is proposed that the kinetic alpha,beta-chain nonequivalence in Hb4(O2)4 is due to the difference in the tension in the two chains on Fe by the proximal base. The absence of alpha,beta-chain differences large enough to show up in CO dissociation rates from Hb4(CO)4 is explained on the basis of lack of sensitivity of the Fe-CO bound to tension from the proximal base. The implications of the results for the observed cooperative effects in ligand combination (for CO) and dissociation (for O2 and NO) rates of hemoglobin have also been discussed.
Sickle cell anemia and trait in a population of southern India.
In an ethnic group in southern India, the Irula, seven individuals with sickle cell anemia were found to manifest only mild illness. Although a relatively high level of fetal hemoglobin was present in one, none of the factors thought to ameliorate the course of sickling disorders could be identified in the remaining six. In a random population survey, sickle hemoglobin was found in 90 of 292 Irula (31%). In those with sickle cell trait, the proportion of sickle hemoglobin in hemolysates (mean = 26%, range 19-32%) was substantially lower than that reported for any other population.
Dissociation of CO from carboxyhemoglobin.
The reaction between carboxyhemoglobin and reduced microperoxidase (MP): Hb4(CO)4 + 4MP=Hb4 + 4MPCO, recently reported by us, has been further studied. By generating species Hb4(CO), Hb4(CO)2, and Hb(CO)3 in the stopped flow cuvette by the reaction of dithionite with the species of the general formula Hb4(O2)x(CO)y(x + y=4) in the presence of microperoxidase it has been possible to determine the stepwise CO dissociation rate constants l4, l3, l2, and l1. The overall CO dissociation rate constant l, which is the same in this system as l4, is not affected by 2,3-diphosphoglyceric acid. The activation energy of the reaction is 21,400 cal in 15-25 degrees range. The ratio deltal/deltapH is approximately 3 in 6.5 to 7.5 pH range. The kinetic data indicate that, compared to HbO2, the contribution to the cooperativity of the dissociation rate constants of carboxyhemoglobin is greatly reduced. The ligand-dependent differences in the reactions of Hb with CO, O2, and NO suggest that in the combination reactions the ligand plays an active role in the rate-limiting step.
Hemoglobin M equon beta 41 (C7) phenylalanine leads to tyrosine.
A severe hemolytic crisis was observed in a 34-yr-old female of English-Irish extraction following a viral illness treated with acetaminophen. Heinz bodies and heat instability were present only during a transient hemolytic event. A challenge dose of acetaminophen caused no detectable hematologic abnormality. Structural studies of the hemoglobin during hemolysis and again after complete recovery localized the abnormality to tryptic peptide beta Tp-5, and automated sequencing of I 125-labeled beta chains indicated a replacement of phenylalanine (C7) beta 41 by tyrosine. Substitution of the next residue, phenylalanine (CD1) beta 42 by serine (Hb Hammersmith), has resulted in chronic severe Heinz body hemolytic anemia. The lack of chronic anemia in the present disorder may reflect the different relationships of beta41 and beta 42 and/or the similarities in volume and hydrophobicity of tyrosine and phenylalanine. It is suggested that substitution of tyrosine for phenylalanine in Hb Mequon may disturb the critical environment around the heme group and render it susceptible to oxidative denaturation in the presence of infections and/or drugs.
A new method for the determination of ligand dissociation rate constant of carboxyhemoglobin.
Explore the source record for details and available documents.
Effect of urea on the kinetics of ligand combination reactions of hemoglobins A and H, alphaSH chains and myoglobin.
Explore the source record for details and available documents.
A comparison of the functional properties of two lepore hemoglobins with those of hemoglobin A1.
Explore the source record for details and available documents.
Rat haemoglobin heterogeneity. Two structurally distinct alpha chains and functional behaviour of selected components.
Six haemoglobins were separated analytically from haemolysates of adult Wistar rats (Rattus norvegicus) by cellulose acetate electrophoresis and preparatively by DEAE-cellulose chromatography. The globin chains were separated from unfractionated haemolysates by CM-cellulose chromatography by using a non-linear formic acid-pyridine gradient followed by CM-cellulose chromatography in 8M-urea by using a gradient of increasing Na+ concentration in phosphate buffer, pH 6.7. Two alpha chains and three non-alpha chains were identified. Chains isolated from purified haemoglobins were correlated with chains isolated from unfractionated haemolysates by electrophoresis on urea-starch gels to make presumptive assignments of the subunit composition of the six haemoglobin tetramers. Partial amino acid sequences were determined for the major and minor alpha chains. The oxygen equilibria of two of the major haemoglobin components and of the unfractionated haemolysate were examined at pH 7.5 and 8.0. The two purified haemoglobins exhibited similar oxygen affinities; the haemolysate, however, had a lower oxygen affinity than either of the two purified haemoglobins. Both the haemolysate and the two haemoglobins showed an alkaline Bohr effect larger than that of human haemoglobin A.