NMR determination of enantiomers of 7-chloro-3,3a-dihydro-2-methyl-2H,9H-isoxazolo(3,2-b)(1,3)benzoxazin-9-one using chiral shift reagent, tris(3-(heptafluorobutyryl)-d-camphorato)europium(III).

A simple NMR method was developed for the determination of the enantiomers of 7-chloro-3,3a-dihydro-2-methyl-2H,9H-isoxazolo[3,2-b][1,3]benzoxazin-9-one. Chiral shift reagent, tris[3-(heptafluoroburyryl)-d-camphorato]europium(III), causes the doublet assigned to the protons of the 2-methyl group, which normally appears at about 1.5 ppm, to split into two pairs of doublets and to shift downfield to about 2.0-3.5 ppm. The downfield pair of doublets represents the two enantiomers present in one racemate, designated as the beta-form, while the upfield pair represents the enantiomers of the racemate designated as the alpha-form. From the integration of the area under the doublets, the relative concentration of all four enantiomers was determined.

Equilibrium binding of alkyl isocyanides to human hemoglobin.

The reactions of human hemoglobin with a series of 13 alkyl isocyanides have been examined in equilibrium titration experiments at pH 7, 20 degrees C. The ligands include: methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, (+)- and (-)-sec-butyl, tert-butyl, n-pentyl, n-hexyl, cyclohexyl, and benzyl isocyanides. All of these compounds exhibit sigmoidal binding curves; however, the amount of cooperativity expressed decreases with ligand length and increases as the alkyl side chains become more highly substituted. The overall affinity of hemoglobin for these compounds also exhibits a complex dependence on ligand size and stereochemistry. These results have been interpreted in terms of competing favorable hydrophobic interactions and unfavorable protein steric effects. The overall chemical potentials of the bound ligands were calculated from the sum of the observed binding free energy change and the relative chemical potentials of the isonitriles in aqueous solution. The resulting values allowed the construction of a rough, three-dimensional free energy map of steric hindrance at the sixth coordination position of the heme iron atom. This scheme suggests a cylindrical cavity of weak protein interactions into which ethyl isocyanide can easily fit. This cavity or mobile region of protein structure is surrounding by a more rigid region which results in large unfavorable steric interactions. Finally, this ring of more rigid structure is followed by an outer area where considerably smaller steric hindrance effects are observed.

Kinetic and cooperative mechanisms of ligand binding to hemoglobin.

The reactions of 13 isonitriles with deoxyhemoglobin have been examined and characterized at pH 7, 20 degrees C. Kinetic studies have shown that these ligands can be divided into two mechanistic classes based on their size and stereochemistry. The larger and branched compounds (isopropyl, all butyl isomers, n-pentyl, n-hexyl, cyclohexyl, and benzyl isocyanides) exhibit biphasic time courses at all ligand concentrations as a result of intrinsic differences between the reactivities of the alpha and beta subunits. In contrast, the smaller isonitriles (methyl, ethyl, and n-propyl isocyanides) exhibit monophasic, and often accelerating, time courses at high ligand concentrations. At low concentrations, the smaller isonitriles also exhibit biphasic time courses; however, in this case, the two phases are due to marked differences between the dissociation rate constants of the high and low affinity quaternary conformations of the protein. Sets of equilibrium and kinetic data for the binding of 11 of the isonitriles were fitted to an expanded version of the two-state allosteric model first described by Monad, Wyman, and Changeux (Monod, J., Wyman, J., and Changeux, J.-P. (1965) J. Mol. 12, 108 118). The resultant rate and equilibrium constants for the R (high affinity) and T (low affinity) states were used to calculate chemical potentials for ligand molecules bound to the heme iron and for the kinetic barriers experienced by these compounds during the binding process. For the beta subunits, both the difference between the bound chemical potentials for the R and T states and the differences between the barrier potentials for the two protein conformations are independent of ligand length and stereochemistry. Thus, it would appear that steric interactions are not a major factor in the expression of cooperativity by these subunits. For alpha chains, a 30% decrease in the difference between the bound R and T state potentials is observed in going from methyl to n-hexyl isocyanide. In addition, a marked increase in the height of the T state, alpha chain kinetic barrier is observed with increasing length of the alkyl side chain. Thus, steric hindrance between the bound ligand molecule and protein residues at the sixth coordination position of the heme iron atom does appear to play a significant role in the expression of cooperativity by the alpha subunits, at least for the larger alkyl isocyanides.

Aggregation of deoxyhemoglobin subunits.

The formation of deoxyhemoglobin was examined by measuring the heme spectral change that accompanies the aggregation of isolated alpha and beta chains. At low hemeconcentrations (less than 10(-5) M), tetramer formation can be described by two consecutive, second order reactions representing the aggregation of monomers followed by the association of alphabeta dimers. At neutral pH, the rates of monomer and dimer aggregation are roughly the same, approximately 5 X 10(5) M(-1) X(-1) at 20 degrees. Raising or lowering the pH results in a uniform decrease of both aggregation rates due presumably to repulsion of positively charged subunits at acid pH and repulsion of negatively charged subunits at alkaline pH. Addition of p-hydroxymercuribenzoate to alpha chains lowers the rate of monomer aggregation whereas addition of mercurials to the beta subunits appears to lower both the rate of monomer and the rate of dimer aggregation. At high heme concentrations (greater than 10(-5) M) or in the presence of organic phosphates, the rate of chain aggregation becomes limited, in part, by the slow dissociation of beta chain tetramers. In the case of inositol hexaphosphate, the rate of hemoglobin formation exhibits a bell-shaped dependence on phosphate concentration. When intermediate concentrations of inositol hexaphosphate (approximately 10(-4 M) are preincubated with beta subunits, a slow first order time course is observed and exhibits a half-time of about 8 min. As more inositol hexaphosphate is added, the chain aggregation reaction begins to occur more rapidly. Eventually at about 10(-2) M inositol hexaphospate, the time course becomes almost identical to that observed in the absence of phosphates. The increase in the velocity of the chain aggregation reaction at high phosphate concentrations suggests strongly that inositol hexaphosphate binds to beta monomers and, if added in sufficiently large amounts, promotes beta4 dissociation. A quantitative analysis of these results showed that the affinity of beta monomers for inositol hexaphosphate is the same as that of alphabeta dimers. Only when tetramers are formed, either alpha2beta2 or beta4, is a marked increase in affinity for inositol hexaphosphate observed.

Kinetic resolution of ligand binding to the alpha and beta chains within human hemoglobin.

Nitric oxide has been used as a chain-specific, spin label of unliganded heme groups present in kinetic mixtures of human hemoglobin and n-butyl isocyanide. In these experiments, deoxyhemoglobin was reacted with n-butyl isocyanide for a controlled time and then mixed rapidly with a high concentration of nitric oxide to fill residual, unoccupied heme sites. The final mixture was frozen immediately after formation to prevent any displacement of bound isonitrile. The EPR spectrum of the frozen sample was resolved into alpha and beta nitric oxide components; these reflect the relative proportions of alpha- and beta-heme sites which were unoccupied by n-butyl isocyanide. Individual time courses for the alpha and beta subunits were obtained by varying the time between the formation of the isonitrile/hemoglobin mixture and its reaction with nitric oxide. At pH 7.0 only the beta chain time course exhibits an initial rapid phase; the alpha chain time course is monophasic, exhibiting almost, exponential behavior. This result shows unequivocally that the beta-hemes within deoxyhemoglobin react much more rapidly with n-butyl isocyanide than the alpha hemes.