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J S Olson

Publications and source records attributed to J S Olson.

At least 73 records · Page 4Linked to original sources
PubMed1993

The mechanism of autooxidation of myoglobin.

Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37 degrees C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, His64(E7) was replaced with Gln, Gly, Ala, Val, Thr, Leu, and Phe; Val68(E11) was replaced with Ala, Ile, Leu, and Phe; Leu29(B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His64(E7) was replaced with Val; Val68(E11) was replaced with Thr and Ser; Thr67(E10) was replaced with Ala, Val, Glu, and Arg; Lys45(CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo-first order rate constants varied over 4 orders of magnitude, from 58 h-1 (H64A) to 0.055 h-1 (native) to 0.005 h-1 (L29F) at 37 degrees C, pH 7, in air. The dependences of the observed autooxidation rate constant on oxygen concentration and pH were measured for native and selected mutant myoglobins. In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high [O2], direct dissociation of the neutral superoxide radical (HO2) from oxymyoglobin dominates, and this process is accelerated by decreasing pH. At low [O2], autooxidation occurs by a bimolecular reaction between molecular oxygen and deoxymyoglobin containing a weakly coordinated water molecule. The neutral side chain of the distal histidine (His64) inhibits autooxidation by hydrogen bonding to bound oxygen, preventing both HO2 dissociation and the oxidative bimolecular reaction with deoxymyoglobin. Replacement of His64 by amino acids incapable of hydrogen bonding to the bound ligand markedly increases the rate of autooxidation and causes the superoxide mechanism to predominate. Increasing the polarity of the distal pocket by substitution of Val68 with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II).O2 complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 (E11) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.

Animals↗
PubMed1993

Recombinant human hemoglobin: modification of the polarity of the beta-heme pocket by a valine67(E11)-->threonine mutation.

Using the mutagenesis and a gene expression system previously described [Fronticelli et al. (1991) J. Protein Chem. 10, 495-501], we have replaced Val67E11 in the distal heme pocket of the beta-chains of hemoglobin with Thr. The valine to threonine substitution is isosteric and only modifies the polarity of the beta-heme environment. The absorption and CD spectra of the resultant mutant hemoglobin were essentially the same as that of wild-type protein, indicating that the mutation did not cause any large conformational changes and that a water molecule was not coordinated to the ferrous iron atom. Equilibrium measurements of oxygen binding to the mutant indicate a 2-fold decrease in overall affinity relative to native or wild-type human hemoglobin. Thermodynamic analyses of O2 binding curves, based either on the sequential Adair model or on the MWC two-state model, indicated that the overall decrease of O2 affinity in the system was due to a lower association equilibrium constant for the intermediates of oxygenation, particularly those involved at the third ligation step. The functional characteristics of the mutant hemoglobin in either the T- or R-state were not modified greatly by the mutation; however, the Bohr effect and sensitivity to C1- were increased, suggesting a role of the intermediates of oxygenation in the modulation of these parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

HIV infection among secondary school students in Djibouti, horn of Africa: knowledge, exposure and prevalence.

A study of HIV infection among secondary school students was conducted in Djibouti in February 1990 during a national hepatitis survey. Serology was negative for HIV and syphilis among 294 students (ages 14 to 20 years) in spite of a dramatic increase of HIV seropositivity (as high as 41%), and a high level of syphilis positive serology (up to 46%) among adult high-risk groups such as street prostitutes. Results of a face-to-face interview of these pupils indicated that 64% had a basic knowledge of AIDS. As expected, a much better knowledge of AIDS was found among those who recently received AIDS education. However, 80% of secondary school teenagers did not consider themselves at risk for acquiring HIV. Transmission of HIV among school-children would be possible through sexual activity (22% stated to be sexually active) or parenteral therapy (75% had received at least one intramuscular injection), and not likely through blood transfusion or intravenous drug abuse. Results, close to those of a similar study in Zimbabwe, suggest that AIDS education programme should be given to all secondary school pupils and include public health lectures on syphilis, tuberculosis, and HIV infection.

Adolescent↗
PubMed1992

Distal pocket residues affect picosecond ligand recombination in myoglobin. An experimental and molecular dynamics study of position 29 mutants.

Time courses for intramolecular NO and O2 recombination to native and three position 29 mutants of sperm whale myoglobins were measured after laser photolysis on picosecond and nanosecond time scales. The rates for the first phase of NO recombination were 1.8, 2.5, 29, and > or = 100 ns-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, at room temperature. This order is not correlated with the overall association rate constants for NO binding which were all in the range 20-50 x 10(6) M-1 s-1 and is the opposite of that observed for the rate constants for the overall thermal dissociation of NO which were 5.0, 2.8, 0.98, and 0.21 x 10(-4) s-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, at 20 degrees C. This inverse correlation suggests that photo- and thermally dissociated ligand molecules experience similar kinetic and equilibrium barriers to rebinding. The larger side chains of Leu29 and Phe29 inhibit rapid movement of the ligand away from the iron atom facilitating geminate recombination. The smaller side chains of Val29 and Ala29 increase the space available to the ligand, decreasing the rate of geminate recombination and enhancing complete dissociation. Diffusion of NO in the distal pocket of myoglobin was simulated using a variant of the molecular dynamics program CHARMM that includes the locally enhanced sampling protocol (Elber, R., and Karplus, M. (1991) J. Am. Chem. Soc. 112, 9161-9175; Roitberg, A., and Elber, R. (1991) J. Chem. Phys. 95, 9277-9287) and the x-ray structures of Carver et al. (Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, M. L., Phillips, G. N., Jr., and Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450). Both accelerated (5,000 K) and room temperature ligands were used, and comparisons were made between simulations with a complete hydration shell surrounding the protein and those with only eight water molecules near the distal histidine. Photodissociated ligands initially move away from the heme plane, past Leu29, and toward Leu32, Phe33, Ile107, and Ile111. These theoretical results confirm that a complete description of picosecond ligand recombination must include the dynamics of ligand movement in the distal portion of the heme pocket.

Animals↗
PubMed1992

A novel site-directed mutant of myoglobin with an unusually high O2 affinity and low autooxidation rate.

Mutants of sperm whale myoglobin were constructed at position 29 (B10 in helix notation) to examine the effects of distal pocket size on the rates of ligand binding and autooxidation. Leu29 was replaced with Ala, Val, and Phe using the synthetic gene and Escherichia coli expression system of Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 8961-8965). Structures of the ferric forms of Val29 and Phe29, and the oxy form of Phe29 myoglobin were determined to 1.7 A by x-ray crystallography. The ferric mutant proteins are remarkably isomorphous with the wild type protein except in the immediate vicinity of residue 29. Thus, the protein structure in the distal pocket of myoglobin can accommodate either a large "hole" (i.e. Ala or Val) or a large side chain (i.e. Phe) at position 29 without perturbation of tertiary structure. Phe29 oxymyoglobin is also identical to the native oxy protein in terms of overall structure and interactions between the bound O2 and His64, Val68, Phe43, and Ile107. The distance between the nearest side chain atom of residue 29 and the second atom of the bound oxygen molecule is 3.2 A in the Phe29 protein and 4.9 A in native myoglobin. The equilibrium constants for O2 binding to Ala29, Val29, and Leu29 (native) myoglobin are the same, approximately 1.0 x 10(6) M-1 at 20 degrees C, whereas that for the Phe29 protein is markedly greater, 15 x 10(6) M-1. This increase in affinity is due primarily to a 10-fold decrease in the O2 dissociation rate constant for the Phe29 mutant and appears to be the result of stabilizing interactions between the negative portion of the bound O2 dipole and the partially positive edge of the phenyl ring. Increasing the size of residue 29 causes large decreases in the rate of autooxidation of myoglobin: k(ox) = 0.24, 0.23, 0.055, and 0.005 h-1 for Ala29, Val29, Leu29 (native), and Phe29 myoglobin, respectively, in air at 37 degrees C. Thus, the Leu29----Phe mutation produces a reduced protein that is remarkably stable and is expressed in E. coli as 100% MbO2. The selective pressure to conserve Leu29 at the B10 position probably represents a compromise between reducing the rate of autooxidation and maintaining a large enough O2 dissociation rate constant to allow rapid oxygen release during respiration.

Amino Acid Sequence↗
PubMed1991

The assignment of carbon monoxide association rate constants to the alpha and beta subunits in native and mutant human deoxyhemoglobin tetramers.

The association kinetics of CO binding to site-directed mutants of human deoxyhemoglobin were measured by stopped-flow rapid mixing techniques at pH 7.0, 20 degrees C. Hemoglobin tetramers were constructed from one set of native subunits and one set of mutated partners containing His(E7) to Gly, Val(E11) to Ala, or Val(E11) to Ile substitutions. The reactivity of beta Cys93 with p-hydroxymercuribenzoate was measured to ensure that the mutant deoxyhemoglobins were capable of forming T-state quaternary conformations. Time courses for the complete binding of CO were measured by mixing the deoxygenated proteins with a 5-fold excess of ligand in the absence and presence of inositol hexaphosphate. Association rate constants for the individual alpha and beta subunits in the T-state conformation were assigned by measuring time courses for the reaction of a small, limiting amount of CO with a 20-fold excess of deoxyhemoglobin (i.e. Hb4 + CO----Hb4(CO)). The effects of the E7 and E11 mutations in T-state alpha subunits were qualitatively similar to those observed for the same subunit in the R-state (Mathews, A.J., Rohlfs, R.J., Olson, J.S., Tame, J., Renaud, J-P., and Nagai, K. (1989) J. Biol. Chem. 264, 16573-16583). The alpha His58(E7) to Gly and Val62(E11) to Ala substitutions caused 80- and 3-fold increases, respectively, in k'CO for T-state alpha subunits, and the alpha Val62(E11) to Ile mutation caused a 3-fold decrease. The beta His63(E7) to Gly and Val67(E11) to Ala substitutions produced 70- and 8-fold increases, respectively, in k'CO for T-state beta subunits whereas these same mutations caused little effect on the rate of CO binding to R-state beta subunits. The beta Val67(E11) to Ile mutation produced the same large effect, a 23-fold reduction in k'CO, in both quaternary conformations of beta subunits. These kinetic results can be interpreted qualitatively in terms of differences between the alpha and beta subunits in the deoxy and liganded crystal structures of human hemoglobin (Perutz, M.F. (1990) Annu. Rev. Physiol. 52, 1-25). Both the structural and functional data suggest that the distal portion of the beta heme pocket is tightly packed in deoxyhemoglobin whereas the CO binding site in R-state beta subunits is much more open. In contrast, the distal portion of the alpha heme pocket is restricted sterically in both quaternary states.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1991

Determination of the rate and equilibrium constants for oxygen and carbon monoxide binding to R-state human hemoglobin cross-linked between the alpha subunits at lysine 99.

The kinetics of O2 and CO binding to R-state human hemoglobin A0 and human hemoglobin cross-linked between the alpha chains at Lys99 residues were examined using ligand displacement and partial photolysis techniques. Oxygen equilibrium curves were measured by Imai's continuous recording method (Imai, K. (1981) Methods Enzymol. 76, 438-449). The rate of the R to T transition was determined after full laser photolysis of the carbon monoxide derivative by measuring the resultant absorbance changes at an isosbestic point for ligand binding. Chemical cross-linking caused the R-state O2 affinity of alpha subunits to decrease 6-fold compared with unmodified hemoglobin. This inhibition of O2 binding was the result of both a decrease in the rate constant for ligand association and an increase in the rate constant for dissociation. The O2 affinity of R-state beta subunits was reduced 2-fold because of an increase in the O2 dissociation rate constant. These changes were attributed to proximal effects on the R-state hemes as the result of the covalent cross-link between alpha chain G helices. This proximal strain in cross-linked hemoglobin was also expressed as a 5-fold higher rate for the unliganded R to T allosteric transition. The fourth O2 equilibrium binding constant, K4, measured by kinetic techniques, could be used to analyze equilibrium curves for either native or cross-linked hemoglobin. The resultant fitted values of the Adair constants, a1, a2, and a3 were similar to those obtained when K4 was allowed to vary, and the fits were of equal quality. When K4 was fixed to the kinetically determined value, the remaining Adair constants, particularly a3, became better defined.

Carbon Monoxide↗
PubMed1991

Contributions of residue 45(CD3) and heme-6-propionate to the biomolecular and geminate recombination reactions of myoglobin.

Overall association and dissociation rate constants were measured at 20 degrees C for O2, CO, and alkyl isocyanide binding to position 45 (CD3) mutants of pig and sperm whale myoglobins and to sperm whale myoglobin reconstituted with protoheme IX dimethyl ester. In pig myoglobin, Lys45(CD3) was replaced with Arg, His, Ser, and Glu; in sperm whale myoglobin, Arg45(CD3) was replaced with Ser and Gly. Intramolecular rebinding of NO, O2, and methyl isocyanide to Arg45, Ser45, Glu45, and Lys45(native) pig myoglobins was measured following 35-ps and 17-ns excitation pulses. The shorter, picosecond laser flash was used to examine ligand recombination from photochemically produced contact pairs, and the longer, nanosecond flash was used to measure the rebinding of ligands farther removed from the iron atom. Mutations at position 45 or esterification of the heme did not change significantly (less than or equal to 2-fold) the overall association rate constants for NO, CO, and O2 binding at room temperature. These data demonstrate unequivocally that Lys(Arg)45 makes little contribution to the outer kinetic barrier for the entry of diatomic gases into the distal pocket of myoglobin, a result that contradicts a variety of previous structural and theoretical interpretations. However, the rates of geminate recombination of NO and O2 and the affinity of myoglobin for O2 were dependent upon the basicity of residue 45. The series of substitutions Arg45, Lys45, Ser45, and Glu45 in pig myoglobin led to a 3-fold decrease in the initial rate for the intramolecular, picosecond rebinding of NO and 4-fold decrease in the geminate rate constant for the nanosecond rebinding of O2. (ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Analysis of the kinetic barriers for ligand binding to sperm whale myoglobin using site-directed mutagenesis and laser photolysis techniques.

Time courses for NO, O2, CO, methyl and ethyl isocyanide rebinding to native and mutant sperm whale myoglobins were measured at 20 degrees C following 17-ns and 35-ps laser excitation pulses. His64 (E7) was replaced with Gly, Val, Leu, Phe, and Gln, and Val68 (E11) was replaced with Ala, Ile, and Phe. For both NO and O2, the effective picosecond quantum yield of unliganded geminate intermediates was roughly 0.2 and independent of the amino acids at positions 64 and 68. Geminate recombination of NO was very rapid; 90% rebinding occurred within 0.5-1.0 ns for all of the myoglobins examined; and except for the Gly64 and Ile68 mutants, the fitted recombination rate parameters were little influenced by the size and polarity of the amino acid at position 64 and the size of the residue at position 68. The rates of NO recombination and ligand movement away from the iron atom in the Gly64 mutant increased 3-4-fold relative to native myoglobin. For Ile68 myoglobin, the first geminate rate constant for NO rebinding decreased approximately 6-fold, from 2.3 x 10(10) s-1 for native myoglobin to 3.8 x 10(9) s-1 for the mutant. No picosecond rebinding processes were observed for O2, CO, and isocyanide rebinding to native and mutant myoglobins; all of the observed geminate rate constants were less than or equal to 3 x 10(8) s-1. The rebinding time courses for these ligands were analyzed in terms of a two-step consecutive reaction scheme, with an outer kinetic barrier representing ligand movement into and out of the protein and an inner barrier representing binding to the heme iron atom by ligand occupying the distal portion of the heme pocket. Substitution of apolar amino acids for His64 decreased the absolute free energies of the outer and inner kinetic barriers and the well for non-covalently bound O2 and CO by 1 to 1.5 kcal/mol, regardless of size. In contrast, the His64 to Gln mutation caused little change in the barrier heights for all ligands, showing that the polar nature of His64 inhibits both the bimolecular rate of ligand entry into myoglobin and the unimolecular rate of binding to the iron atom from within the protein. Increasing the size of the position 68(E11) residue in the series Ala to Val (native) to Ile caused little change in the rate of O2 migration into myoglobin or the equilibrium constant for noncovalent binding but did decrease the unimolecular rate for iron-O2 bond formation.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1990

Transmembrane movement of heme.

Evidence for CO-heme partitioning into and across lipid bilayers was obtained by kinetic and chromatographic studies. Biphasic time courses were observed when CO-heme was rapidly mixed with unilamellar lipid vesicles in a stopped-flow spectrometer. The initial rapid phase depended linearly on lipid concentration and was assigned to heme partitioning between the external solvent phase and the outer lipid layer of the membranes. The rate of the second, much slower phase was independent of both heme and lipid concentration. The fraction of absorbance change associated with this slower phase increased with increasing heme to lipid ratios and reached a maximum of approximately 45%. A similar slow phase was observed when membrane-bound heme was reacted with apomyoglobin. In the presence of excess globin, all of the CO-heme was extracted from the membranes to form native CO myoglobin. Under these conditions, the fractional amount of absorbance change associated with the slow dissociation phase was approximately 45%, regardless of the heme to lipid ratio. These results suggest strongly that the slow phases represent transmembrane movement of heme, from the outer to the inner lipid layer in the association reactions and from the inner to the outer layer in dissociation reactions. The temperature dependence of the rate of CO-heme binding to the outer lipid layer was markedly different from that of transmembrane movement. The rate of the latter, slower process decreased greatly with increasing acyl chain length, whereas the rate of the initial binding process varied little with vesicle composition, as long as the membranes were examined above their melting temperatures. Finally, the two kinetically distinct bound heme fractions could be isolated directly by column chromatography.

Apoproteins↗
PubMed1990

The effects of lipid composition on the rate and extent of heme binding to membranes.

The effects of membrane composition on heme binding to large unilamellar vesicles were examined using 30 separate phospholipid mixtures. Although there was some variation, most lecithins with Tm values less than or equal to 20 degrees C showed overall equilibrium partition constants equal to approximately 5 x 10(5) and association and dissociation partition rate constants equal to approximately 3 x 10(6) s-1 and 7 s-1, respectively, for CO-heme binding at 30 degrees C. A sharp decrease in the association rate for CO-heme uptake was observed as the lipid vesicles changed from liquid-crystalline to the gel phase. The addition of dicetyl phosphate or dimyristoylphosphatidylglycerol, which are negatively charged at neutral pH, decreased the affinity of the vesicles for CO-heme. The association rate and equilibrium partition constants for CO-heme uptake in unsaturated lecithins were unaffected by cholesterol content at levels up to 40%/mol. The affinity of saturated dimyristoylphosphatidylcholine (DMPC) vesicles for CO-heme decreased with increasing cholesterol content at 30 degrees C. This effect appears to be related to the influence of cholesterol on the DMPC phase transition temperature (Tm) since at low temperatures (less than or equal to 20 degrees C) little CO-heme binds to vesicles composed of DMPC even in the absence of cholesterol.

Carbon Monoxide↗
PubMed1990

The role of Val68(E11) in ligand binding to sperm whale myoglobin. Site-directed mutagenesis of a synthetic gene.

Site-directed mutants of sperm whale myoglobin were prepared to probe the functional role of the highly conserved distal pocket valine residue, Val68(E11). This amino acid was replaced with Ala, Ile, and Phe to examine the effects of the side chain volume at position 68 on ligand binding. Three double mutants were also constructed in which the distal His64(E7) was replaced with Gly and Val68 was replaced with Ala, Ile, and Phe to determine the effects of size at position 68 in the absence of the distal histidine. Association and dissociation rate constants for O2, CO, and alkyl isocyanide binding were measured by stopped-flow rapid mixing, conventional flash, and laser photolysis techniques at pH 7, 20 degrees C. The association rate constants for the binding of all eight ligands to the single mutants decreased in the order Ala68 greater than Val68 (native) greater than Ile68 myoglobin, indicating that the 68(E11) residue is part of the overall kinetic barrier. A similar pattern was observed for the association constants of the double mutants: Gly64/Ala68 greater than Gly64/Val68 greater than Gly64/Ile68. Thus, increasing size of the E11 side chain inhibits the rate of ligand binding even in the absence of histidine at position 64. Substitution of Ala for Val68 had little effect on O2 affinity but did increase the affinities for CO and isocyanide binding. The affinities for all of the ligands were decreased for the Ile68 mutant. The ligand binding affinities for the Gly64/Ala68, Gly64/Val68, and Gly64/Ile68 myoglobins displayed an analogous trend to that of the single mutants, indicating that the equilibrium interactions between the position 64 and 68 side chains and the bound ligand are roughly additive. Both the association rate constants and dissociation rate constants for O2 and isocyanide binding were decreased for the Phe68 mutant myoglobin. These kinetic parameters result in little change in O2 affinity and an increase in isocyanide affinity, relative to the native protein. Thus, the large benzyl side chain of phenylalanine at position 68 inhibits the rate of ligand movement up to and away from the iron atom but not the final bound state.

Amino Acid Sequence↗
PubMed1990

The effects of amino acid substitution at position E7 (residue 64) on the kinetics of ligand binding to sperm whale myoglobin.

Association and dissociation rate constants were measured for O2, CO, and alkyl isocyanide binding to a set of genetically engineered sperm whale myoglobins with site-specific mutations at residue 64 (the E7 helical position). Native His was replaced by Gly, Val, Leu, Met, Phe, Gln, Arg, and Asp using the synthetic gene and expression system developed by Springer and Sligar (Springer, B. A., and Sligar, S. G. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8961-8965). The His64----Gly substitution produced a sterically unhindered myoglobin that exhibited ligand binding parameters similar to those of chelated protoheme suspended in soap micelles. The order of the association rate constants for isocyanide binding to the mutant myoglobins was Gly64 (approximately 10(7) M-1 s-1) much greater than Val64 approximately Leu64 (approximately 10(6) M-1 s-1) greater than Met64 greater than Phe64 approximately His64 approximately Gln64 (10(5)-10(3) M-1 s-1) and indicates that the barrier to isocyanide entry into the distal pocket is primarily steric in nature. The bimolecular rates of methyl, ethyl, n-propyl, and n-butyl isocyanide binding to the His64----Arg and His64----Asp mutants were abnormally high (1-5 x 10(6) M-1 s-1), suggesting that Arg64 and Asp64 adopt conformations with the charged side chains pointing out toward the solvent creating a less hindered pathway for ligand binding. In contrast to the isocyanide data, the association rate constants for O2 and CO binding exhibited little dependence on the size of the E7 side chain. The values for all the mutants except His64----Gln approached or were larger than those for chelated model heme (i.e. approximately 1 x 10(8) M-1 s-1 for O2 and approximately 1 x 10(7) M-1 s-1 for CO), whereas the corresponding rate parameters for myoglobin containing either Gln64 or His64 were 5- to 10-fold smaller. This result suggests that a major kinetic barrier for O2 and CO binding to native myoglobin may involve disruption of polar interactions between His64 and water molecules found in the distal pocket of deoxymyoglobin. Finally, the rate and equilibrium parameters for O2 and CO binding to the His64----Gln, His64----Val, and His64----Leu mutants were compared to those reported previously for Asian elephant myoglobin (Gln-E7), Aplysia limacina myoglobin (Val-E7), and monomeric Hb II from Glycera dibranchiata (Leu-E7).

Amino Acids↗
PubMed1990

A simple model for prediction of oxygen transport rates by flowing blood in large capillaries.

A simple model has been developed for simulation of oxygen transport to and from blood flowing in conduits of the diameter of arterioles and larger (greater than or equal to 20 microns). The basis is the large capillary model (LCAP) of P.K. Nair, et al. 1989 which has been validated experimentally. Detailed calculations of the oxygen concentration distribution reveal that the dominant resistance to oxygen transport is distributed in the plasma. Relatively little resistance is present within or in the immediate vicinity of the red cells. On the basis of these findings, LCAP was simplified from four simultaneous nonlinear partial differential equations (PDEs) to one PDE by (1) assuming chemical equilibrium within the red blood cells, (2) neglecting intracellular and extracellular boundary layer resistances, and (3) incorporating transport in the cell-free region adjacent to the capillary wall into the boundary conditions. The simplified model is much easier to apply mathematically to new situations. A comparison between LCAP and the simpler model shows that they give virtually the same predictions, and the predictions agree well with experimental measurements. The model is predictive in that all the parameters are determined from the literature or from independent measurements. Thus it should be useful in studies of physiological significance, as well as in design and analysis of extracorporeal blood oxygenators.

Biological Transport, Active↗
PubMed1989

The effects of E7 and E11 mutations on the kinetics of ligand binding to R state human hemoglobin.

Association and dissociation rate constants for O2, CO, and methyl isocyanide binding to native and distal pocket mutants of R state human hemoglobin were measured using ligand displacement and partial photolysis techniques. Individual rate constants for the alpha and beta subunits were resolved by comparisons between the kinetic behavior of the native and mutant proteins. His-E7 was replaced with Gly and Gln in both alpha and beta subunits and with Phe in beta subunits alone. In separate experiments Val-E11 was replaced with Ala, Leu, and Ile in each globin chain. The parameters describing ligand binding to R state alpha subunits are sensitive to the size and polarity of the amino acids at positions E7 and E11. The distal histidine in this subunit inhibits the bimolecular rate of binding of both O2 and CO, sterically hinders bound CO and methyl isocyanide, and stabilizes bound O2 by hydrogen bonding. The Val-E11 side chain in alpha chains also appears to be part of the kinetic barrier to O2 and CO binding since substitution with Ala causes approximately 10-fold increases in the association rate constants for the binding of these diatomic ligands. However, substitution of Val-E11 by Ile produces only small decreases in the rates of ligand binding to alpha subunits. For R state beta subunits, the bimolecular rates of O2 and CO binding are intrinsically large, approximately 2-5-fold greater than those for alpha subunits, and with the exception of Val-E11----Ile mutation, little affected by substitutions at either the E7 or E11 positions. For the beta Val-E11----Ile mutant the association rate and equilibrium constants for all three ligands decreased 10-50-fold. All of these results agree with Shaanan's conclusions that the distal pocket in liganded beta subunits is more open whereas in alpha subunits bound ligands are more sterically hindered by adjacent distal residues (Shaanan, B. (1983) J. Mol. Biol. 171, 31-59). In the case of O2 binding to alpha subunits, the unfavorable steric effects are compensated by the formation of a hydrogen bond between the nitrogen atom of His-E7 and bound dioxygen.

Antisickling Agents↗
PubMed1989

Structure of myoglobin-ethyl isocyanide. Histidine as a swinging door for ligand entry.

The structure of myoglobin(Fe II)-ethyl isocyanide has been solved at 1.68 A resolution by X-ray crystallography. The isocyano group of the ligand is distorted from the linear conformation observed in solution and in model compounds. Local changes in the protein conformation are also seen. The side-chain of Arg-CD3 moves out into the solvent, and the side-chain of His-E7 swings up and away from the ligand. Both of these side-chains show disorder indicative of dynamic behavior. These outward movements of His-E7 and Arg-CD3 side-chains clear a path from the solvent to the heme iron, suggesting a mechanism for ligand entry.

Animals↗
PubMed1989

Discrimination between oxygen and carbon monoxide and inhibition of autooxidation by myoglobin. Site-directed mutagenesis of the distal histidine.

Sperm whale myoglobin mutants were constructed using site-directed mutagenesis to replace the highly conserved distal histidine residue (His(E7)-64). His-64 was substituted with Gly, Val, Phe, Cys, Met, Lys, Arg, Asp, Thr, and Tyr, and all 10 mutant proteins expressed to approximately 10% of the total soluble cell protein in Escherichia coli as heme containing myoglobin. With the exception of His-64----Tyr, which did not form a stable oxygen (O2) complex, all mutant proteins could be reduced and bound O2 and carbon monoxide (CO) reversibly. However, removal of the distal histidine increased the rate of autooxidation 40-350-fold. The His-64----Gly, Val, Phe, Met, and Arg mutants all showed markedly increased O2 dissociation rate constants which were approximately 50-1500-fold higher than those for wild-type myoglobin and increased O2 association rate constants which were approximately 5-15-fold higher than those for the native protein. All mutants studied (except His-64----Tyr) showed approximately 10-fold increased CO association rates and relatively unchanged CO dissociation rates. These altered O2 and CO association and dissociation rate constants resulted in 3-14-fold increased CO affinities, 10-200-fold decreased O2 affinities, and 50-380-fold greater M (KCO/KO2) values for the mutants compared to the wild-type protein. Thus, the distal histidine of myoglobin discriminates between CO and O2 binding by both sterically hindering bound CO and stabilizing bound O2 through hydrogen bonding. The increased autooxidation rates observed for the mutants appear to be due to a decrease in oxygen affinity and an increase in solvent anion accessibility to the distal pocket.

Amino Acid Sequence↗
PubMed1989

Prediction of oxygen transport rates in blood flowing in large capillaries.

A mathematical model has been developed to predict oxygen transport to and from blood flowing in tubes of the diameter of arterioles and larger (approximately 20 microns and larger). The resistance to oxygen transport in red cell suspensions is much higher than that of a comparable homogeneous hemoglobin solution. The increased resistance is associated with encapsulation of the hemoglobin in the red cells. Yet, somewhat paradoxically, for large capillaries relatively little resistance is within or in the immediate vicinity of the red cells. The great majority of the resistance is shown to be distributed in the plasma. Predictions of oxygen uptake and release are shown to be in excellent agreement with results of measurements taken on red cell suspensions flowing in capillaries of 27- and 100-microns diameter. The model seems to be the first for oxygen transport in flowing blood that is validated by detailed comparison with experimental results. It is a predictive model in that all parameters in the model are determined from independent measurements or from the literature.

Arterioles↗