Craig M. Jackson

Solution composition dependent variation in extinction coefficients for p-Nitroaniline

The dependence of the extinction coefficients for para-nitroaniline on solution composition has been investigated. The p-nitroaniline absorption spectrum is red-shifted with increasing ionic strength, with the consequence that the extinction coefficients at fixed wavelengths may vary significantly. The isosbestic wavelength for peptide p-nitroanilide/p-nitroaniline mixtures is similarly shifted. Poly(ethylene glycol) and bovine albumin, two additives frequently employed to eliminate enzyme loss from adsorption to cuvette and dilution vessel surfaces, also induce shifts in the p-nitroaniline spectrum. The use of a difference extinction coefficient at 381 nm, the p-nitroaniline maximum wavelength, is proposed to minimize the error resulting from solution composition dependent spectral shifts.

Activation of prothrombin with oxyuranus scutellatus scutellatus (taipan snake) venom

Abstract Complete activation of prothrombin with taipan snake ( Oxyuranus scutellatus scutellatus ) venom yields thrombin and two fragments, fragment-1 and fragment-2, which by the criteria of ion-exchange chromatography and SDS gel electrophoresis are indistinguishable from the products of Xa-catalyzed prothrombin activation. Activation in the presence of diisopropylfluorophosphate with either taipan venom or Xa results in the formation of thrombin-sensitive fragment 1·2, a single polypeptide composed of fragments-1 and 2. Whereas the rate of venom-catalyzed activation is enhanced 20–30 times by phospholipid vesicles, Factor V is without effect in either the presence or absence of phospholipid. The venom activity is not inhibited by diisopropylfluorophosphate, phenylmethylsulfonylfluoride, soybean trypsin inhibitor, p-hydroxymercuribenzoate or iodoacetate. The venom does not hydrolyze N2-toluene-p-sulfonyl-L-arginine methyl ester, N2-benzoyl-L-arginine ethyl ester or N-acetyl-L-tyrosine ethyl ester. The rate of venom-catalyzed prothrombin activation is not affected by plasma factor Xa inhibitor plus heparin.

Cooperative calcium binding by the phospholipid binding region of bovine prothrombin: a requirement for intact disulfide bridges.

Abstract The Fragment 1 portion of bovine prothrombin binds 10 moles of calcium per mol of polypeptide with an isotherm which shows positive cooperativity. The Hill plot derived from the binding data has a slope of 1.50 and the 10 binding sites are 50% saturated at a Ca 2+ concentration of 0.63 m m in 0.05 m Tris-HCl, pH 8.0 and 0.1 m NaCl. Reduction of the four disulfide bridges in Fragment 1 eliminates the cooperativity of the Ca 2+ binding and reduces the average affinity of the polypeptide for Ca 2+ by a factor of 20. Similar experiments on bovine blood clotting Factor X also show positive cooperativity in binding 20 mol of Ca 2+ per mol of native protein with loss of cooperativity and diminished affinity for Ca 2+ upon disulfide reduction. Separate Ca 2+ binding determinations on each of the two isolated chains of Factor X suggest that the Ca 2+ binding capacity of this protein is a property of the smaller, light polypeptide chain.

The action of thrombin on blood clotting factor V: conversion of factor V to a prothrombin-binding protein

Abstract Incubation of “native” factor V with thrombin (EC 3.4.4.13) or the purified factor V-activating enzyme from Russells viper venom converts factor V to a form which binds to a prothrombin-Sepharose affinity column. The thrombin-altered factor V is quantitatively eluted from the affinity column by increasing the ionic strength of the buffer.

The action of thrombin on peptide p-nitroanilide substrates: Hydrolysis of Tos-Gly-Pro-Arg-pNA and D-Phe-Pip-Arg-pNA by human α and γ and bovine α and β-thrombins

Human and bovine α-thrombins (>90% a form) with high fibrinogen clotting activities (∼3,000 U.S. units/mg protein) exhibit similar Michaelis Menten kinetics with the p-nitroanilide tripeptide substrates Tos-Gly-Pro-Arg-pNA (Chromozym-TH) and D-Phe-Pip-Arg-pNA (S-2238). The kinetic parameters at I = 0.11 M, 25°C, pH 7.8 are: (Km = 4.18 ± 0.22 and 3.61 ± 0.15 μM; kcat = 127 ± 8 and 100 ± 1 s−1) for Chromozym TH and (Km = 1.33 ± 0.07 and 1.58 ± 0.10 μM; kcat = 91.4 ± 1.8 and 98.0 ± 0.5 s−1) for S-2238 for the human and bovine enzymes, respectively. Unlike the native enzyme forms, their “non-clotting” terminal degradative forms, human γ-thrombin (∼5 units/mg) and bovine β-thrombin (∼200 units/mg), give increased values for these parameters (Km = 14.3 ± 2.4 and 14.4 ± 2.2 μM; kcat = 160 ± 9 and 124 ± 6 s−1) for Chromozym-TH; and (Km = 2.50 ± 0.36 and 2.99 ± 0.33 μM; kcat = 106 ± 3 and 106 ± 3 s−1) for S-2238. Based on these parameters, 50% degradation of human or bovine α-thrombins can be calculated to produce relatively small errors in the kinetic measurement of total thrombin concentrations (maximally 9% and 7% for Chromozym-TH; 7% and 3% for S-2238, respectively) if the kinetic parameters for all a forms are erroneously used and assays are at 150 μM substrate. This is in contrast to the large errors inherent in clotting activity measurements on thrombin mixtures. Incorporation of 1 mg/ml of polyethylene glycol 6,000 into assay solutions eliminates systematic errors otherwise caused by thrombin adsorption to surfaces and enables thrombin to be accurately assayed at concentrations <0.1 μM or 0.01 clotting unit/ml of α-thrombin.

Phase changes and mosaic formation in single and mixed phospholipid monolayers at the oil-water interface☆

Abstract Insoluble monolayers of phosphatidylcholines and ethanolamines show first-order phase transitions at hydrocarbon-water interfaces, depending on the temperature and chain length. In mixed monolayers of two phosphatidylcholines of different chain lengths, or of phosphatidylcholines and ethanolamines of the same chain length, demixing occurs at the phase transition.

Separation of the tosyl arginine esterase activity from the factor X activating enzyme of Russell's Viper venom

Abstract The coagulant protein activity and the N-p- toluenesulfonyl- l -arginine methyl ester hydrolase activity present in the “coagulant protein” preparation of Vipera russelli have been separated by chromatography on DEAE cellulose. The coagulant protein activity is not inhibited by diisopropyl phosphorofluoridate or phenylmethyl sulfonyl fluoride, whereas the ester hydrolase is inhibited by both of these reagents. These experiments indicate that the coagulant and ester hydrolase activities are catalyzed by separate proteins.

The factor V activating enzyme of Russell's viper venom☆

Abstract The factor V activator of Russells viper venom is an arginine esterase and can be inhibited by diisopropylphosphorofluoridate. It has been shown that this enzyme accounts for one half the total coagulant activity of crude Russells viper venom. The extent of activation of factor V by the venom factor V activator has been found to be the same as obtained using thrombin. In contrast to thrombin, however, the factor V activator neither clots fibrinogen nor does it catalyze proteolysis of prothrombin.

Phospholipid monolayers at the hydrocarbon-electrolyte interface. The interrelation of film potential and film pressure

Measurements of surface pressure of surface potential are reported for films of distearoyl phosphatidylcholine (density range: 0.15--2.65 . 10(18) molecules/m2) spread at the interface between 2,2,4-trimethylpentane and 100 mM NaCl. Low density behavior of the surface pressure is explained using classical viral theory. The behavior of the surface potential is qualitatively explained for all densities in terms of the dipole moments associated with the carboxyl groups and headgroups of the phosphatidylcholine.

Measurement of phospholipid monolayer surface potentials at a hydrocarbon-electrolyte interface.

The phospholipid monolayer spread at a hydrocarbon-electrolyte interface can be used as a model system for the plasma membrane and its properties and structure probed by measurements of surface pressure and surface potential. To facilitate such studies, (i) the theory of the vibrating plate (Kelvin) method of measuring surface potentials is reëxamined and a new interpretation given for the potentials measured and (ii) a new apparatus for performing these measurements is described. The theory and apparatus are illustrated by measurements on films of distearoyl phosphatidylcholine at the interface between 2,2,4-trimethylpentane (isooctane) and 0.1 M NaCl.