Richard G. Hiskey

The reaction of 4,4′-bis-dimethylaminodiphenylcarbinol with the sulfhydryl group: A new reagent for sulfhydryl analysis

Abstract 4,4′-bis-Dimethylaminodiphenylcarbinol (BDC-OH) dissociates in aqueous buffers at pH values below neutrality to form a resonance-stabilized carbonium-immonium ion (BDC+) which exhibits an absorbance maximum at 606 nm. In the presence of 4.0 M guanidine hydrochloride, BDC+ has an apparent molar absorption coefficient of 70,800 M−1cm−1 and an absorbance maximum of 612 nm. Sulfhydryl groups react with the cation to form S-(4,4′-bis-dimethylaminodiphenylmethyl-) derivatives with a concomitant quantitative loss of the 612-nm absorbance. This quantitative interaction has been exploited in the development of a new and convenient technique for the quantitative determination of sulfhydryl groups in proteins. Results of sulfhydryl determinations on simple thiols and five proteins are presented, along with comparison data obtained via other sulfhydryl techniques.

pH Dependence of magnesium ion binding to prothrombin fragment 1 and γ-carboxyglutamic acid-containing peptides via 25MG NMR

Abstract The binding of magnesium ions to two tripeptides, L -Arg- D -Gla- D -Gla-OMe and Z- L -Arg(NO2)- D -Gla- D -Gla-OMe, and to bovine prothrombin fragment 1 as a function of pH has been monitored by 25Mg NMR spectroscopy. Binding to the tripeptide was dependent on peptide ionizations occurring at pH 4.6 – 4.8. The pH dependence of magnesium ion binding to fragment 1 reveals two inflection points 4.2 may be attributed to the deprotonation of the third side chain carboxylic acid group of the double γ-carboxyglutamic acid sequence. The origin of the increased binding of magnesium ions to fragment 1 at pH values above 7 is unknown.

Relative affinity of Ca(II) and Mg(II) ions for human and bovine prothrombin and fragment 1

Equilibrium dialysis results are presented for Ca(II) and Mg(II) ion binding to human and bovine prothrombin and fragment 1. Ca(II) ions bind cooperatively, Mg(II) does not.

Analysis of γ-carboxyglutamic acid via amino acid analysis

Abstract Modifications of the analysis of protein-bound residues of γ-carboxyglutamic acid (Gla) via alkaline hydrolysis are presented. The method described allows easier sample manipulation than that heretofore required and insures quantitative recovery of hydrolyzed amino acids. A possible explanation of the shoulder which sometimes appears near Gla on some amino acid analyzers is presented.

The Role of Hydrated Divalent Metal Ions in the Bridging of Two Anionic Groups. An ab initio Quantum Chemical and Molecular Mechanics Study of Dimethyl Phosphate and Formate Bridged by Calcium and Magnesium Ions

Ab initio quantum chemical (Gaussian82) and molecular mechanics (AMBER2.0) computational techniques are employed to investigate the interaction of two anions (formate an dimethylphosphate) and a central divalent metal cation (magnesium or calcium). These systems are models for the essential GDP binding unit of the G-proteins (e.g., EF-Tu or the ras oncogene proteins) and for protein/phospholipid interactions, both of which are mediated by divalent metal cations. Various levels of hydration are utilized to examine coordination of differences between magnesium and calcium ions. Two different orientations of formate and dimethyl phosphate in direct ion contact with a magnesium ion and two waters of hydration were energy minimized with both quantum and molecular mechanics techniques. The structures and energy differences between the two orientations determined by either of the computational techniques are similar. Magnesium ion has a strong propensity to assume six coordination whereas calcium ion preferentially assumes a coordination greater than six. Likewise, water molecules attached to magnesium ion are held more rigidly than those of calcium ion, thus calcium ion is more accommodating in the exchange of water for negative ligands.

A method for specific chemical modification of γ-carboxyglutamic acid residues in proteins

Abstract A method for the chemical modification of γ-carboxyglutamic acid (Gla) residues in proteins is introduced that has the combined advantages of mildness, a high degree of specificity, and the ability to introduce a radiolabel at modification sites for ease in quantitation. Unlike other Gla modification procedures which are performed in the lyophilized state at 110°C, this procedure is carried out in solution at 37°C. The addition of morpholine and formaldehyde to a slightly acidic solution of bovine prothrombin fragment 1 (residues 1–156) results in the conversion of Gla residues to γ-methyleneglutamic acid (γ-MGlu). The extent of modification is controlled by the relative amounts of modification reagents to protein. A 100-fold molar excess of reagents to fragment 1 produced a protein molecule containing two γ-MGlu residues, while a modification run at 10,000-fold molar excess of reagents to protein yielded fragment 1 containing eight γ-MGlu residues per molecule. The specificity of this modification is illustrated by the interaction of native and modified protein with antibody populations directed against fragment 1. Native fragment 1, 8 γ-MGlu fragment 1, and 2 γ-MGlu fragment 1 show fairly similar behavior toward whole anti-fragment 1 serum. Differential behavior was exhibited by the native and modified proteins toward a subpopulation of antibodies specific to the calcium ion conformation of fragment 1. Unmodified fragment 1 displayed a strong affinity for these antibodies; however, the 2 γ-MGlu fragment 1 exhibited a moderate affinity and the 8 γ-MGlu fragment 1 did not bind to these antibodies. This is in agreement with the current understanding of vitamin K-dependent coagulation proteins which links calcium-dependent behavior to the presence of Gla residues.

Determination of strontium binding to macromolecules.

An equilibrium dialysis technique for determining the binding of strontium to macromolecules is described. The major difficulty to be overcome is that 90Sr has a decay product, 90Y, which is also a beta-emitter. The described protocol is used to determine the Sr binding isotherm to bovine prothrombin fragment 1. The binding is found to be cooperative, somewhat weaker than Ca binding, and to involve approximately nine strontium sites. The stoichiometric equilibrium constants are determined by nonlinear regression. The procedure should be of great utility for many macromolecules that show strontium affinity.

The determination of a calcium-dependent binding constant of the bovine prothrombin GLA domain (residues 1–45) to phospholipid vesicles

Calcium-mediated binding of the radioiodinated peptide representing residues 1-45 of bovine prothrombin to single bilayer phospholipid vesicles composed of phosphatidylserine from bovine brain and synthetic 1-palmitoyl-2-oleoyl-phosphatidylcholine (25:75 PS/PC) has been studied over peptide concentrations from 0.33 microM to 3.75 microM and at a calcium concentration of 1.0 mM. The binding isotherm for the interaction between the radioiodinated peptide and PS/PC vesicles fits a model in which there is noncooperative binding of the peptide to non-interacting sites on the phospholipid bilayer. A dissociation constant determined at these conditions is 11.8 microM compared to 1.0 microM for prothrombin fragment 1.

Inactivation of human blood coagulation factor X by chemical modification of gamma-carboxyglutamic acid residues☆

The inactivation of human factor X by incubation with a reagent known to chemically modify gamma-carboxyglutamic acid to gamma-methylene glutamic acid was studied. Incubation of factor X at pH 5.0 with a preincubated formaldehyde/morpholine mixture (0.9 M/1.0 M) resulted in a progressive decrease in factor X coagulant activity. In the presence of calcium (20 mM) the rate of factor X inactivation was decreased -3-fold. By using [14]C-formaldehyde, modified-factor X (less than 5% residual activity) was found to contain 7 mols of [14]C per mol of protein. Modified-factor X was not activated by Russells viper venom in the presence of calcium, suggesting that the loss of coagulant activity was related to the inability of modified-factor X to be activated.

Chiral synthesis of L-γ-carboxyglutamic acid (L-Gla)

A two step synthesis involving the use of a chiral template, benzyl (2R, 3S)-(−)-6-oxo-2,3-diphenyl-4-morpholine-carboxylate3 (5a), provides orthogonally protected L-Gla (9) in 60% overall yield (>99% ee), with no resolution required.