PubMed Health⌕ Search

Biomedical subjects

R G Hiskey

Publications and source records attributed to R G Hiskey.

At least 55 records · Page 3Linked to original sources
PubMed1983

Dye binding probes of lipid-binding structures. An investigation of 2-p-toluidinylnaphthylene-6-sulfonate binding to human and bovine prothrombin and fragment 1 in the presence and absence of calcium and magnesium ions.

TNS (2-p-toluidinylnaphthylene-6-sulfonate) binds to human and bovine prothrombin and Fragment 1 in the absence and presence of added Ca2+. The stoichiometry of TNS binding is 1:1 for human and bovine prothrombin and Fragment 1. The Ca2+-dependence of the fluorescence of TNS bound to bovine prothrombin Fragment 1 yields a modified Hill plot slope of 2.7, which is consistent with the slope obtained by monitoring the Ca2+ dependence of protein fluorescence quenching, CD changes and phospholipid binding. Mg2+ has have no effect on the fluorescence of TNS-prothrombin fluorescence. TNS binding to the amino-terminal region of prothrombin is the first relatively simple probe of the subtle and complex relationship which exists between protein structure and phospholipid binding.

Animals↗
PubMed1981

Europium(III) binding to bovine prothrombin residues 1-39 and to bovine prothrombin fragment 1.

Changes in the Eu3+ luminescence decay constant observed for Eu3+ bound to prothrombin fragment 1 and the peptide containing residues 1-39 of prothrombin result solely from changes in the number of water molecules in the primary hydration sphere of the ion. Highly coordinated sites which bind Eu3+ in a different manner from simple Gla-containing peptides exist on both the fragment 1 and 1-39 molecules. Metal ions bound at some of these sites do not appear to undergo rapid exchange with metal ions in solution. The binding of other lanthanide ions and calcium ions in the presence of europium ions cause a change in the conformation of the macromolecules, resulting in even tighter coordination of fragment 1 or residues 1-39 to Eu3+. Hydrophilic groups other than the actual Eu3+ binding sites on fragment 1 help to maintain its water solubility when complexes to Eu3+ or Ca2+. Because the 1-39 peptide does not contain as many of these hydrophilic groups, the peptide precipitates upon binding to di- and trivalent cations. Self-association of the 1-39 molecules appears to occur at high peptide concentrations which result in a peptide concentration effect on Eu3+ binding not seen in fragment 1. pH titration yields results which suggest a role for Gla carboxyl groups in Eu3+ complexation to both fragments 1 and 1-39. Although the two molecules are not identical with regard to Eu3+ binding behavior, enough similarities exist that residues 1-39 appear to be a reasonable model of the fragment 1 molecule for the purposes of metal ion complexation studies.

Amino Acid Sequence↗
PubMed1981

Kinetic and equilibrium metal-ion-binding behaviour reflected in a metal-ion-dependent antigenic determinant in bovine prothrombin. Comparison with bovine prothrombin fragment 1.

Rabbit anti-(bovine prothrombin fragment 1) antibodies were fractionated by using fragment-1 affinity chromatography in the absence of metal ions, and showed an absolute requirement for the presence of metal ions in their interactions with bovine fragment 1 or prothrombin. These antibodies were employed to evaluate both the rate constants for a protein conformation change and the equilibrium metal-ion binding to isolated bovine fragment 1 and intact prothrombin. The close similarity of the rates obtained for the conformation change in fragment 1 and those observed in prothrombin indicated that the same process is involved in both proteins and that the non-fragment-1 region of the prothrombin has essentially no effect on this process in the fragment-1 region. Equilibrium metal-ion-binding studies indicate that the details of the metal-ion-binding process in fragment 1 and prothrombin are essentially the same. We conclude that the metal-ion-binding behaviour of the fragment-1 domain of intact prothrombin is identical with that of isolated fragment 1.

Animals↗
PubMed1981

Tryptophan exposure in various conformational isomers of bovine prothrombin fragment 1. An acrylamide quenching study.

In order to assess the importance of a variety of environmental factors on the structure of bovine prothrombin fragment 1, we have examined acrylamide quenching of fragment 1 intrinsic fluorescence. Tryptophan exposure, determined from Stern-Volmer plots, is heterogeneous with one or more of the three fragment 1 tryptophans being exposed to solvent. In the presence of Ca2+ or Mg2+ even the most accessible tryptophan(s) are relatively buried. Only small differences in tryptophan exposure may exist between fragment 1-Ca2+ and fragment 1-Mg2+ complexes. Lowering pH, on the other hand, results in increased tryptophan exposure. Finally, structural isomers of fragment 1 which exist in the absence of metal ions have identical tryptophan exposure as determined by acrylamide quenching and fluorescence intensity.

Acrylamides↗
PubMed1980

Bovine prothrombin fragment 1, segment 1-10. Synthesis and immunological investigation.

The N-terminal decapeptide methyl ester, H-Ala-Asn-Lys-Gly-Phe-Leu-Gla-Gla-Val-Arg-OCH3 (16) of bovine prothrombin fragment 1 has been prepared by standard solution techniques, via a fragment coupling strategy. Hexapeptide Boc-Ala-Asn-Lys epsilon (Boc)-Gly-Phe-Leu-OBzl (9) was obtained by coupling Boc-Ala-Asn-Lys epsilon (Boc)Gly-OH (6) to the trifluoroacetate salt of H-Phe-Leu-OBzl (8). Hydrogenolysis of (9) followed by coupling to HCl. H-Gla gamma (OtBu)2-Gla gamma (OtBu)2-Val-Arg(HCl)-OCH3 (14) gave the fully protected decapeptide (15). Treatment of 15 with 90% trifluoroacetic acid followed by ion exchange chromatography of 15 yielded the methyl ester (16). The decapeptide 16 labeled with 125I using the Bolton-Hunter reagent, did not bind to antibodies specific for the calcium ion-induced conformation of bovine fragment 1.

Amino Acid Sequence↗
PubMed1979

The nature of the slow metal ion-dependent conformational transition in bovine prothrombin.

Kinetic parameters characterizing the slow structural isomerization observed via metal ion-dependent intrinsic fluorescence quenching of bovine prothrombin Fragment 1 have been determined. From forward and reverse rate constants, an equilibrium constant of approx. 0.25 is calculated. This result is consistent with the hypothesis that there exists, in the absence of metal ions, an equilibrium between two forms of bovine Fragment 1, one of which can interact rapidly with Ca2+ and subsequently with phospholipid. The other form of Fragment 1 cannot interact with Ca2+ in a manner that yields a phospholipid-binding form of the protein. Interconversion of these two forms of Fragment 1 occurs and may involve the isomerization of a proline residue.

Animals↗
PubMed1979

Structure of a Gla-containing dipeptide. The crystal structure of (+/-)-N-carbobenzoxy-(gamma,gamma'-DI-tertbutyl)-gamma-carboxyglutamylglycine ethyl ester.

The crystal and molecular structure of a dipeptide containing a blocked gamma-carboxyglutamyl (Gla) residue is presented. Two intermolecular hydrogen bonds link the amides with carbonyl groups in the dipeptide backbone, but the protected gamma-carboxy groups on the modified glutamic acid are not hydrogen bonded.

1-Carboxyglutamic Acid↗
PubMed1979

Physical studies on isolated human prothrombin fragment-2. Comparisons with human prothrombin fragment-1.

Variation of pH strongly affects the fluorescence intensity of human prothrombin fragment-1 in a manner suggesting contributions from a number of protropic equilibria including groups with apparent pKa values near 3.0. These results suggest a structural role for pK1a of gamma-carboxyglutamic acid noieties. Added calcium ions (9 mM calcium chloride) quench the fluorescence titration curve uniformly above pH 4. Below pH 4, however, the titration curve in the presence of calcium ions suggests that calcium-ion-dependent processes leading to fluorescence quenching are pH-dependent. Upon back titration of human fragment-1, from pH 9, hysteresis is observed. Human prothrombin fragment-2 fluorescence titration curves are relatively broad at low pH suggesting the titration of normal carboxyl groups. The titration curves of fragment-2 are not affected by the presence of calcium ions, and hysteresis occurs upon back titration from low pH values. Circular dichroism (CD) Cotton effects appear at 232 nm and 280 nm and a trough appears at 203 nm in the CD spectrum of human prothrombin fragment-2. The Cotton effects in the region from 230 nm to 300 nm are sensitive to pH, ellipticity values at 232 nm increasing from approximately 300 at pH 2.5 to 1300 (degree-cm/decimole) at neutral pH and finally become negative at high pH values. In contrast to fragment-1, at neutral pH the fragment-2 Cotton effect at 232 nm is insensitive to the presence of 8 mM calcium chloride.

Calcium↗
PubMed1979

The effects of calcium ions and pH on bovine prothrombin fragment 1. Intrinsic fluroescence studies.

The effects of pH and Ca2+ on the intrinsic fluorescence of bovine prothrombin fragment 1 were investigated to deduce the nature of protein functional groups involved in Ca2+ binding to fragment 1. From pH values of 9 to 3, increasing the H3O+ concentration results in quenching of the fluorescence of fragment 1. Reversible pH-titration curves are obtained which appear to consist of two regions. From pH 4 to pH6.5 a broad titration curve is obtained, whereas from pH6.5 to 9 a more pronounced titration behaviour is evidenced by a group or groups on fragment 1 with an apparent pKa of approx. 7.5. In contrast, the apparent association constant for Ca2+ and fragment 1 shows a sharp pH-dependence in the region between pH7 and 8 with tighter Ca2+ binding at higher pH values. A PKa of approx. 7.5 can be estimated for the group or groups on fragment 1 linked to the tight binding of Ca2+. Both H3O+ and Ca2+ result in blue-shifts in the wave-lengths of fragment-1 emission. These results are interpreted in terms of H+ - and Ca2+ - induced changes in the conformation of fragment 1 as a result of surface-charge neutralization.

Animals↗
PubMed1978

Calcium and magnesium binding to gamma-carboxyglutamic acid-containing peptides via metal ion nuclear magnetic resonance.

The determination of binding constants of metal ions to biomolecules is approachable via many techniques. Metal ion NMR spectroscopy is an alternative to more traditional techniques and is complementary to them, particularly in investigations of the interactions of metal ions with relatively small peptides containing multiple ionizing groups. The method requires relatively small amounts of material, is fairly fast, and is carried out at equilibrium. Our study has been of calcium and magnesium ion binding to gamma-carboxyglutamic acid (Gla)-containing peptides. Dissociation constants of approximately 0.6 mM for the binding of either metal ion to Z-D-Gla-D-Gla-OMe have been obtained. The procedure for determination of these constants via metal ion NMR is discussed.

Calcium↗
PubMed1978

Spin-labeled ribonuclease A. Effects of chemical, enzymatic, and physical modifications on enzyme conformation.

3-SLHis-105-RNase A is an active derivative of ribonuclease A (RNase A) spin-labeled at the 3 position of the imidazole ring of histidine-105. The spin-labeled enzyme has been modified by urea denaturation, reduction, reduction-carboxymethylation, performic acid oxidation, and digestion with proteolytic enzymes in order to monitor changes in the geometry of the protein by changes in the electron paramagnetic resonance (EPR) spectrum of the nitroxide spin-label probe. The results of these experiments indicate that the spin-label attached to histidine-105 of RNase A is sensitive to modifications affecting the conformational integrity of the molecule and to the reconstituting effects of various active-center ligands.

Animals↗