Biomedical subjects
Publications and source records attributed to D W Deerfield.
An equilibrium dialysis technique for examining magnesium binding to macromolecules is described. The technique is used to determine the binding constants of magnesium to human prothrombin. This procedure should be of great utility for many biochemical systems which exhibit magnesium affinity.
A unique method is presented for the determination of the critical number of ligands that must bind to a macromolecule to elicit a spectroscopic response. This method is based on analysis of ligand binding data. For example, four Ca2+ and two Mg2+ ions are necessary for mirroring the relative decrease in the intrinsic fluorescence of bovine prothrombin fragment 1. For application of the method, ligand loading and relative spectroscopic response data must be measured over a full range of concentrations.
The formaldehyde-morpholine method for the conversion of gamma-carboxyglutamyl (Gla) residues to gamma-methyleneglutamyl (gamma-MGlu) residues has been applied to the modification of bovine prothrombin fragment 1. In the absence of Tb3+ ions or at Tb3+ ion concentrations of 2 Km app and 25 Km app the action of 10,000-fold molar excess of formaldehyde and morpholine, pH 5.0, converts the 10 Gla residues of the protein into 10 gamma-MGlu residues. Modification of the protein using the same conditions but increasing the Tb3+ concentration to 100 Km app provided a homogeneous protein containing 3 gamma-MGlu and 7 Gla residues, bovine 3 gamma-MGlu-fragment 1. The modified protein binds the same number of Ca2+ ions (6-7) as bovine fragment 1. However, the positive cooperatively associated with Ca2+ binding is abolished and the overall affinity for Ca2+ ions is reduced. Fluorescence titrations of 3 gamma-MGlu-fragment 1 using either Ca2+ or Mg2+ ions indicate that the modified protein retains a fluorescence quenching behavior similar to that of the native protein. The modified protein does not bind to phosphatidylserine/phosphatidylcholine vesicles in the presence of Ca2+ ions. Thus the metal ion-induced fluorescence transition exhibited by the bovine protein appears to be a necessary but not sufficient condition for phospholipid binding.
Examination of metal ion-dependent effects on the electrophoretic mobility of bovine prothrombin and fragment 1 provides a useful and sensitive method for investigation of conformational processes in these proteins. Utilization of this method reveals a conformational change in bovine prothrombin and fragment 1 which occurs at low metal ion concentrations. Equilibrium dialysis studies indicate that the metal ion-induced shape change occurs concomitant with binding of a single calcium ion/molecule of prothrombin or fragment 1. Mixed metal electrophoretic mobility studies with Mg2+ and Ca2+ have demonstrated the "synergistic" effect for fragment 1 observed by others. Mixed metal equilibrium dialysis has provided experimental support for this observation and allows us to conclude that two tight Ca2+ sites are not affected by low Mg2+ concentrations and that the third Ca2+ site is also a tight site for Mg2+. Thus, at low Mg2+ concentrations and upon the addition of Ca2+, there are effectively three tight sites; consequently more Ca2+ will bind to the protein at lower total Ca2+ ion concentrations.
The vitamin K-dependent proteins involved in blood coagulation processes each contain a small cystine loop with the sequence -Gla-Cys-X-Gla-Gla-X-Cys-. A method for the synthesis of the heptapeptide derivative representing the 17-23 sequence of bovine prothrombin, Z-Gla-Cys-Leu-Gla-Gla-Pro-Cys-OBzl, has been developed. The 25Mg2+ n.m.r. spectrum of the heptapeptide derivative has been obtained and is compared to the n.m.r. spectra obtained from the interaction of 25Mg2+ with Z-Gla-OMe and Z-Gla-Gla-OMe.