Circular dichroism as a probe of the allosteric R in equilibrium T transformation in hemoglobins and modified hemoglobins.

The circular dichroism spectra at pH 6.5 of a number of hemoglobins and modified hemoglobins have been recorded in the 280 nm region and interpreted in terms of shifts of the R?T allosteric transformation. Inositol hexaphosphate converts aquomet hemoglobin A(S) to the T form but the carbamlyated derivatives are unaffected by inositol hexaphosphate and remain in the R form. Fluorodinitrobenzene and dimethyl adipimidate modified hemoglobins are locked in an intermediate form, and inositol hexaphosphate has little or no effect. The circular dichroism in the 280 nm region is shown to be a useful diagnostic tool for chemical agents that affect the R?T allosteric transformation.     

Functional properties of partially oxidized trout hemoglobins.

This paper reports on a study of the effect of partial oxidation on oxygen and carbon monoxide binding by components I and IV of trout hemoglobin. The O2 binding equilibria of the various oxidation mixtures show a decrease in the heme-heme interactions as the number of oxidized sites is increased. However, the large Bohr effect, characteristic of Hb Trout IV, is maintained unchanged. Similarly the time course of CO combination changes on increasing the fractional oxidation, and the autocatalytic character of the CO binding kinetics is lost; however the pH dependence of the apparent "on" constant in the oxidation mixtures is similar to that characteristic of the native molecule. The results of the O2 equilibria and of CO binding kinetics may be interpreted in accordance with the two state concerted model suggesting that in the oxidation intermediates there is an increase in the fraction of the high affinity (R) conformation. Additional experiments on the effect of azide, and fluoride, ferric ligands which produce a change of spin state of the heme iron, suggest that additional second order conformational changes may also come into play.     

Functional and conformational properties of mouse hemoglobins

Hemoglobins from four strains of mice (C3H/SW, DBA/2J, C57BL6/Kh and A.TH) examined showed pH-dependent heme-heme interactions. The oxygen affinity and cooperativity are reduced at acidic pH. The oxygen equilibrium parameters increase as a function of increasing pH and at physiological pH values they are similar to the corresponding values of human hemoglobin A. The nitrosyl derivatives of these mouse hemoglobins undergo a quaternary structural transition to the T state in going from pH 7.0 to 6.0. These functional and conformational properties are indicative of destabilised oxy structures of mouse hemoglobins at acidic pH. This study also confirms that the cysteine residue at beta 13(A10) position has no influence on the oxygen equilibrium properties or conformation of the molecule.     

Functional properties of human hemoglobins synthesized from recombinant mutant beta-globins.

The previous and following articles in this issue describe the recombinant synthesis of three mutant beta-globins (beta 1 Val----Ala, beta 1 Val----Met, and the addition mutation beta 1 + Met), their assembly with heme and natural alpha chains into alpha 2 beta 2 tetramers, and their X-ray crystallographic structures. Here we have measured the equilibrium and kinetic allosteric properties of these hemoglobins. Our objective has been to evaluate their utility as surrogates of normal hemoglobin from which further mutants can be made for structure-function studies. The thermodynamic linkages between cooperative oxygenation and dimer-tetramer assembly were determined from global regression analysis of multiple oxygenation isotherms measured over a range of hemoglobin concentration. Oxygen binding to the tetramers was found to be highly cooperative (maximum Hill slopes from 3.1 to 3.2), and similar patterns of O2-linked subunit assembly free energies indicated a common mode of cooperative switching at the alpha 1 beta 2 interface. The dimers were found to exhibit the same noncooperative O2 equilibrium binding properties as normal hemoglobin. The most obvious difference in oxygen equilibria between the mutant recombinant and normal hemoglobins was a slightly lowered O2 affinity. The kinetics of CO binding and O2 dissociation were measured by stopped-flow and flash photolysis techniques. Parallel studies were carried out with the mutant and normal hemoglobins in the presence and absence of organic phosphates to assess their allosteric response to phosphates. In the absence of organic phosphates, the CO-binding and O2 dissociation kinetic properties of the mutant dimers and tetramers were found to be nearly identical to those of normal hemoglobin. However, the effects of organic phosphates on CO-binding kinetic properties of the mutants were not uniform: the beta 1 + Met mutant was found to deviate somewhat from normalcy, while the beta 1 Val----Met mutant reproduced the native allosteric response. Further characterization of the allosteric properties of the beta 1 Val----Met mutant was made by measuring the pH dependence of its overall oxygen affinity by tonometry. Regulation of oxygen affinity by protons was found to be nearly identical to normal hemoglobin from pH 5.8 to 9.3 (0.52 +/- 0.07 protons released per oxygen bound at pH 7.4). The present study demonstrates that the equilibrium and kinetic functional properties of the recombinant beta 1 Val----Met mutant mimic reasonably well those of normal hemoglobin. We conclude that this mutant is well-suited to serve as a surrogate system of normal hemoglobin in the production of mutants for structure-function studies.     

Copper and the oxidation of hemoglobin: a comparison of horse and human hemoglobins.

Oxidation studies of hemoglobin by Cu(II) indicate that for horse hemoglobin, up to a Cu(II)/heme molar ratio of 0.5, all of the Cu(II) added is used to rapidly oxidize the heme. On the other hand, most of the Cu(II) added to human hemoglobin at low Cu(II)/heme molar ratios is unable to oxidize the heme. Only at Cu(II)/heme molar ratios greater than 0.5 does the amount of oxidation per added Cu(II) approach that of horse hemoglobin. At the same time, binding studies indicate that human hemoglobin has an additional binding site involving one copper for every two hemes, which has a higher copper affinity than the single horse hemoglobin binding site. The Cu(II) oxidation of human hemoglobin is explained utilizing this additional binding site by a mechanism where a transfer of electrons cannot occur between the heme and the Cu(II) bound to the high affinity human binding site. The electron transfer must involve the Cu(II) bound to the lower affinity human hemoglobin binding site, which is similar to the only horse hemoglobin site. The involvement of beta-2 histidine in the binding of this additional copper is indicated by a comparison of the amino acid sequences of various hemoglobins which possess the additional site, with the amino acid sequences of hemoglobins which do not possess the additional site. Zn(II), Hg(II), and N-ethylmaleimide (NEM) are found to decrease the Cu(II) oxidation of hemoglobin. The sulfhydryl reagents, Hg(II) and NEM, produce a very dramatic decrease in the rate of oxidation, which can only be explained by an effect on the rate for the actual transfer of electrons between the Cu(II) and the Fe(II). The effect of Zn(II) is much smaller and can, for the most part, be explained by the increased oxygen affinity, which affects the ligand dissociation process that must precede the electron transfer process.     

Functional properties of normal and sickle cell hemoglobins in polyethylene glycol 6000.

The functional properties of human hemoglobin A and S were studied in concentrated solutions of polyethylene glycol. Polyethylene glycol solutions are frequently used as media for protein crystallization. In particular, sickle cell hemoglobin, which does not make X-ray quality crystals in high salt solutions, will form high-quality crystals in polyethylene glycol. Comparison of the functional properties of normal and sickle cell hemoglobin in polyethylene glycol show that pH, anion effects and cooperativity of ligand binding are largely unaffected by polyethylene glycol. This suggests that the crystals grown in this medium are representative of the native structure.     

Human hemoglobin cross-linked through the polyphosphate-binding site. Functional properties and evidence for conformers

The properties of human hemoglobin reacted with 2-nor-2-formylpyridoxal 5'-phosphate, a bifunctional derivative of pyridoxal 5'-phosphate, have been investigated both from an equilibrium and kinetic point of view. The experimental data, interpreted in terms of the two-state allosteric model, indicate that a perturbed R state is characteristic of this modified low ligand affinity hemoglobin. In flash photolysis experiments, a quickly reacting component is always observed, in spite of the lack of dissociation into free dimers; this kinetic behavior is thought to reflect the presence of functionally independent alpha beta dimers, still connected by the flexible cross-link but forming an open hemoglobin tetramer. Two possible models for the interpretation of the kinetics of CO and/or haptoglobin binding are presented and discussed.     

O(2) affinity of cross-linked hemoglobins modifies O(2) metabolism in proximal tubules.

Previous experiments using cross-linked tetrameric hemoglobins (XLHb) to perfuse isolated rat kidneys showed that high-O2-affinity XLHb improved proximal tubule function more effectively than low-O2-affinity XLHb. To determine how function was improved, proximal tubule fragments were incubated with albumin, Hb34 [half-saturation point (P50) 34 Torr], or Hb13 (P50 13 Torr) with Po2 values ranging from 22 to 147 Torr. ATP content reflected O2 delivery to mitochondria. Both XLHb increased ATP, Hb34 with Po2 >or= 47 Torr and Hb13 with Po2 相似文献   

Evolution of ruminant hemoglobins. Thermodynamic divergence of ox and buffalo hemoglobins.

The ligand-binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed. Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion. In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the delta H for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (delta H = -30.5 kJ/mol O2). This discrepancy with the other ruminant hemoglobins (e.g. ox, delta H = -10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.     

Use of silicate sol-gels to trap the R and T quaternary conformational states of pig kidney fructose-1,6-bisphosphatase.

Encapsulation of the homotetrameric pig kidney fructose-1,6-bisphosphatase (FBPase) in tetramethyl orthosilicate sol-gels was used to dramatically reduce the rate of the allosteric transition of the enzyme between the T and R allosteric states. When assayed in the absence of the allosteric inhibitor AMP, the enzyme encapsulated in the T-state exhibited little activity. The enzyme encapsulated in the R-state exhibited a 4-fold lower k(cat) and V(max) than the enzyme in solution, and the apparent K(m) for this enzyme was 350-fold higher than the corresponding value for the enzyme in solution. The [Mg(2+)](0.5) for the encapsulated enzyme was only 0.1 mM, compared to 0.54 mM for the normal enzyme. Magnesium activation, under both sets of conditions, was cooperative with a Hill coefficient of approximately 2. The activity of enzyme encapsulated in the R-state decreased to about 70% of initial activity within 1 min of adding AMP, it then decreased slowly to about 40% of initial activity over the following 7 h. Under the conditions tested, the encapsulated enzyme never became completely inactivated and AMP inhibition was no longer cooperative. For enzyme encapsulated in the T-state, activity was restored over approximately 7 h after removal of the AMP. The biphasic and slow responses to changing AMP levels suggest that encapsulated enzyme can be used to study the effects of local conformational changes distinct from the global quaternary conformational changes by slowing down the ability of the enzyme to carry out global rotations. The response to AMP exhibited by the encapsulated enzyme is consistent with the ability of AMP, at least partially, to directly influence the activity of the active site within each subunit.     

Stereoselective reactivity of the SH groups of yeast glyceraldehydephosphate dehydrogenase in the allosteric T and R states

Yeast glyceraldehyde-3-phosphate dehydrogenase as a typical SH enzyme is inactivated by the antipodes of a-iodopropionic acid and its amide at different rates. The apoenzyme reacts faster with the D(+) antipode of the free a-iodopropionic acid (k(D)/k(L) = 6.8) and the L(-) antipode of the amide (k(L)/k(D) = 3). On addition of NAD(+) the stereoselectivity of the SH group towards a-iodopropionic acid is inverted, that towards the amide is enlarged, the rate relationships depending on the NAD(+) concentration.The results were interpreted by the assumption, that the allosteric T state of the enzyme reacts most rapidly with the D(+) antipodes, whereas the R state favours the L(-) antipodes of the alkylation reagents. The dependence of the reaction rates on the NAD(+) concentration could be fitted to the allosteric function of state R.     

Interaction of sodium dodecyl sulfate with human native and cross-linked hemoglobins: a transient kinetic study

The interaction of sodium dodecyl sulfate (SDS) at a concentration range (0-515 microM) below the critical micelle concentration (CMC approximately 0.83 mM) with human native and cross-linked oxyhemoglobin (oxyHb) and methemoglobin (metHb) has been investigated by optical spectroscopy and stopped-flow transient kinetic measurements. It is observed that the interaction of SDS with human native and cross-linked oxyHb shows the disappearance of the bands of oxyHb at 541 and 576 nm and the appearance at 537 nm. The resultant spectra are characteristic of low spin (Fe(3+)) hemichrome. Similarly SDS has been found to convert human native and cross-linked high spin (Fe(3+)) metHb to low spin (Fe(3+)) hemichrome. The interaction of SDS with oxyHb suggests a conformational change of the protein in the heme pocket, which may induce the binding of distal histidine to iron leading to the formation of superoxide radical. The formation of hemichrome from metHb is found to be concentration-dependent with SDS. The stopped flow transient kinetic measurements of the interaction of SDS with metHb show that at least four molecules of SDS interact with one molecule of metHb. The interaction of SDS with human cross-linked oxy and met hemoglobin shows results similar to those for human native oxy and met hemoglobin indicating that the covalent modification does not alter the interaction of SDS with cross-linked hemoglobin.     

Binding of acellular, native and cross-linked human hemoglobins to haptoglobin: enhanced distribution and clearance in the rat

It is well established that hemoglobin resulting from red cell lysis binds to haptoglobin in plasma to form a complex. The increased molecular size precludes its filtration by the kidneys, redirecting it toward hepatocellular entry. Chemically cross-linked hemoglobins are designed to be resistant to renal excretion, even in the absence of haptoglobin. The manner in which binding to haptoglobin influences the pharmacokinetics of acellular cross-linked and native hemoglobins was investigated after intravenous injection of radiolabeled native human hemoglobin and trimesyl-(Lys82)beta-(Lys82)beta cross-linked human hemoglobin, at trace doses, into rats. Under these conditions, there is sufficient plasma haptoglobin for binding with hemoglobin. In vitro binding assayed by size-exclusion chromatography for bound and free hemoglobin revealed that, at <8 muM hemoglobin, native human hemoglobin was completely bound to rat haptoglobin, whereas only approximately 30% of trimesyl-(Lys82)beta-(Lys82)beta cross-linked hemoglobin was bound. Plasma disappearance of low doses (0.31 mumol/kg) of native and cross-linked hemoglobins was monoexponential (half-life = 23 and 33 min, respectively). The volume of distribution (40 vs. 19 ml/kg) and plasma clearance (1.22 vs. 0.4 ml.min(-1).kg(-1)) were higher for native than for cross-linked hemoglobin. Native and cross-linked human hemoglobins were found primarily in the liver, and not in the kidney, heart, lung, or spleen, mostly as degradation products. These pharmacokinetic findings suggest that the binding of hemoglobin to haptoglobin enhances its hepatocellular entry, clearance, and distribution.     

Copolymers from unsaturated macrolactones: toward the design of cross-linked biodegradable polyesters

The enzymatic synthesis of a series of random copolyesters by ring-opening polymerization of unsaturated macrolactones like globalide and ambrettolide with 1,5-dioxepan-2-one (DXO) and 4-methyl caprolactone (4MeCL) was investigated. (13)C NMR diad analysis confirmed the randomness of all copolymers irrespective of the comonomer ratios. Thermal investigation showed that incorporating the comonomers lowered the melting points of the polymers as compared with the macrolactone homopolymers. The decrease was dependent on the comonomer ratio. The unsaturated copolymers were thermally cross-linked using dicumyl peroxide, which resulted in completely amorphous insoluble networks. It was found that 10% incorporation of the unsaturated macolactone was sufficient to obtain a gel content of 95 wt %. Preliminary degradation tests confirm that the cross-linked copolymers are enzymatically degradable and that the incorporation of hydrophilic comonomers like DXO enhances degradation.     

Reactivity of chemically cross-linked fibrinogen and its fragments D toward the staphylococcal clumping receptor

It has been established that the binding domain for the staphylococcal clumping receptor exists in fragment D of human fibrinogen [Hawiger J., Timmons, S., Strong, D. D., Cottrell, B. A., Riley, M., & Doolittle, R. F. (1982) Biochemistry 21, 1407; Strong, D. D., Laudano, A., Hawiger, J., & Doolittle, R. F. (1982) Biochemistry 21, 1414]. To examine the role of valency in the adhesive function of fibrinogen, its fragments were prepared by digestion with plasmin in the presence of calcium and purified by a two-step chromatographic procedure. Fragments D1 and E did not induce the staphylococcal clumping reaction. After they were prepared in oligomeric form by chemical cross-linking with glutaraldehyde, fragment D1 (Mr 94,000) became functionally reactive toward the staphylococcal clumping receptor, and fragment D3 (Mr 75,000) and fragment E (Mr 50,000) remained inactive. Fragment D dimer derived from enzymatic cross-linking was not reactive. Human fibrinogen cross-linked with glutaraldehyde usually reached a 250 times higher reactivity toward the staphylococcal clumping receptor, depending on the condition of the cross-linking reaction. It is concluded that the valency of fibrinogen in regard to its receptor binding domain and the availability of this domain are essential for the staphylococcal clumping reaction.