Solvent Ionic Strength Research Articles

Preparation of Highly Purified Prothrombin Complex: I. CRYSTALIZATION, BIOLOGICAL ACTIVITY, AND MOLECULAR PROPERTIES

Abstract Bovine prothrombin, an α2-glycoprotein clotting factor of plasma, has been isolated in high degree of purity in crystalline form as the barium glycoprotein product of interaction. Chemical determinations made on the product showed the following composition, in percentage of the dry weight: protein, 21.5%; barium, 32%; and citrate, 8.9%. That the crystals represent a true prothrombin-metal complex has been established by control studies, x-ray diffraction pattern, chemical composition, and recrystallization. The product showed constant solubility characteristics. Extensive physical and chemical studies of the barium-free protein are presented. Further purification was achieved by gel filtration on Sephadex G-100. The peak fractions evidenced activity of 3000 Iowa units (Seegers, W. H., Prothrombin, Harvard University Press, Cambridge, Massachusetts, 1962, p. 420) per mg (corresponding to a 600-fold purification), as well as appreciable Factors VII, IX, and X coagulant activities. This product is designated prothrombin in accordance with the nomenclature used by other investigators. The preparation was homogeneous by sedimentation velocity pattern, sedimentation equilibrium analysis, and gel filtration. Despite these indications of homogeneity, physical heterogeneity was shown by immuno- and disc electrophoresis. The multiple coagulant activities and protein subcomponents were found to be homogeneous with respect to molecular weight (70,477 ± 2,789), determined by sedimentation equilibrium in a dissociating solvent consisting of 6 m guanidine hydrochloride and 0.5% mercaptoethanol, and also with respect to Stokes radius. Physical constants, determined in various systems, include diffusion constant, sedimentation coefficient, frictional ratio, isoelectric point, isoionic point, electrophoretic mobility, and extinction coefficient. Physical data failed to show evidence of dissociation into subunits or fragmentation by dilution to below 0.1% protein or by prolonged exposure to a dissociating solvent. The latter observation suggests that the reduction in sedimentation constant noted in high ionic strength solvents in this study, and by other observers, is probably attributable to changes in shape and hydration of the kinetic unit. The product was also shown to undergo rapid, reversible association in solvents of low ionic strength (T/2 = 0.15). DEAE-Sephadex chromatography resulted in partial activation of the complex, and several homogeneous protein species were isolated and partially characterized. The monomeric molecular weights determined by sedimentation equilibrium in a solvent of 6 m guanidine hydrochloride and mercaptoethanol were as follows: (devoid of Factors VII and X), 65,530 ± 1,247; modified zymogen, 52,395 ± 3,449; Factor VII, 33,900 ± 3,390; and Factor X, 37,772 ± 1,234. The modified zymogen was inert in the two-stage assay and in 25% sodium citrate, but readily transformed to thrombin when Factor X was added. The results are consistent with the interpretation that the complex, as isolated in this study, comprises a family of glycoproteins that can undergo a proenzyme-enzyme transformation and that have similar molecular properties but separate clotting activities.

Inhibition of the fibrinogen-plasmin reaction by ω-aminocarboxylic acids and alkylamines

Abstract We measured (with the pH stat) the rat of hydrolysis of fibrinogen and lysine methyl ester by plasmin (EC 3.4.4.1.4) and calculated the maximal velocity (vmax) for both reactions. The vmax for the lysine methyl ester-plasmin reaction is 1.5 times greater than the vmax for the fibrinogen-plasmin reaction at pH 7.0, and at 23°. The same enzyme preparation and solvent ionic strength were used in both reactions. A new method for measuring proteolysis of fibrinogen by plasmin is reported. This method is based on the precipitation of non-hydrolyzed fibrinogen by 9% ethanol. With this method we investigated inhibition of the fibrinogen-plasmin reaction by ω-aminocarboxylic acids and analogues of some of the 3,4,5 and 6-carbon members. Glycine, β-alanine, γ-aminobutyric acid, glutaric acid, valeric acid, cadaverine and lysine did not inhibit with concentrations less than 10 mM (Ki>10 mM). Competitive inhibition was found with δ-aminovaleric acid (Ki = 4.7 mM), ϵ-aminocaproic acid (Ki = 2.8 mM), ω-aminocaprylic acid (Ki = 1.0 mM), propylamine (Ki = 3.3 mM)

THE INFLUENCE OF IONIC STRENGTH AND ORGANIC SOLVENTS ON ACID TRANSITIONS OF PROTEINS.

1. 1. The influence of ionic strength, dioxane, and ethylene glycol on the acid transition of ribonuclease has been studied by difference spectrophotometry. In this transition two of the three buried tyrosyl residues of the molecule can be normalized if the temperature is high enough, but an increase in the ionic strength will repress the normalization of one of them. Dioxane makes the normalization of the second one easier, and ethylene glycol has very little effect. 2. 2. Dioxane also raises the p K of the transition. Ribonuclease, serum albumin, and acetic acid all show the same dependence of Δ p K on dioxane concentration, suggesting that the increase is caused by a change in the bulk dielectric constant of the medium. Chymotrypsinogen is much more strongly affected, and lysozyme is less strongly affected. 3. 3. The denaturation of ribonuclease at pH 2.3 by dioxane shows some very unusual features. The difference spectra are anomalous. The denaturation is very strongly affected by the ionic strength, and in an opposite direction to what is usually observed: denaturation occurs at lower dioxane concentrations as the ionic strength is raised. A possible explanation for these unusual features is based on Weber 's observation that dioxane first denatures ribonuclease, and at higher concentrations promotes the formation of helical regions in the molecule.

Light‐scattering studies on sugar beet arabans

AbstractThe discrepancy in the light‐scattering molecular weight of sugar beet araban in two solvent systems, 2M NaCl and 1% (v/v) DMF, is explained as being due to a charge effect in the latter solvent. The previously reported explanation of a multicomponent effect in 1% (v/v) DMF, based on the assumption that araban is uncharged, is shown to be incorrect by equilibrium dialysis experiments. Titration and sodium analysis indicate a total charge of about 20 per araban molecule, while the limiting slope of the Debye plot of light‐scattering data in low ionic strength solvent indicate an effective charge of 10. Attempts to explain the observed curvature in the Debye plot for araban in low ionic strength buffer on the basis of the Stigter‐Hill method was unsatisfactory, probably due to the inadequacy of the solid sphere‐diffuse double layer model for araban and/or the inapplicability of the Stigter‐Hill virial expansion method at high ratios of macromolecule to salt in low ionic strength solvent. Molecular weight measurements on araban acetate prepared for purification purposes indicate considerable degradation during deacetylation but not during acetylation. This suggests that araban in situ has a molecular weight considerably greater than that observed for the purified araban.

Lightweight floating bridge

It has been nearly 60 years since Toms [Toms, B.A., 1949 first discovered the tremendous reduction in wall shear stress caused by the addition of small amount of linear macromolecules to a flowing fluid. This drag reduction phenomenon corresponds to an increase in pumpability of a fluid by the additive. Therefore the application of this phenomenon can be found in many areas of the oil and gas industry; including well completion, workover, drilling operations, and hydraulic fracturing. The turbulent flow prevails in such operations. The eddy currents and velocity fluctuations in turbulent motion produce increased pressure drops and thus, limit the maximum obtainable fluid flow rate. This results in a problem particularly in hydraulic fracturing where higher flow rates are desired. Employing the commercially available drag reducing fluids can overcome this limitation.Salinity and temperature are among the factors that affect the extent of drag reduction. Other factors include polymer type and concentration, molecular weight and distribution, solvent chemistry, pH, ionic strength, molecular conformation, flow geometry, and degree of mechanical shearing. Despite the widespread use of brine solutions as base fluids in fracturing the deep hot formations, the effects of temperature and salinity on the drag reduction characteristics of polymers have received the least attention. Most studies on drag reduction have considered either water or salt-free base fluids at ambient temperatures. Therefore, the aim of the present study is to experimentally investigate the effects of salinity and temperature on the extent of drag reduction of polymers flowing in straight circular pipes.Two commonly used anionic polyacrylamides; Nalco ASP-700 and ASP-820 are used in this study: ASP-700 is an analog of partially hydrolyzed polyacrylamide (PHPA), while ASP-820 is a sulfonate-containing polyacrylamide. A 1/2-in. OD flow loop with a 15-ft straight tubing is used. Flow tests are conducted to determine the optimum concentrations of ASP-700 and ASP-820 polymers. It is found that the optimum concentration for both polymers is 0.07% (vol.). To investigate the effect of temperature, tests are conducted with fresh water at 72, 100 and 130 °F. Additional tests are conducted at ambient temperature and with 2% KCl brine and synthetic sea water to investigate the effect of salinity on drag reduction of ASP-700 and ASP-820.It is found that both polymers are quite effective drag reducers in circular straight pipes. Drag reduction in the range of 30–80% is noticed. Both temperature and salinity have significant effect on their drag reduction characteristics. Due to differences in their composition and potentially molecular weight variances, both polymers show different characteristics in the presence of salt and at elevated temperatures. Within the conditions studied, ASP-700 is found to be better than ASP-820 at providing drag reduction. Correlations to predict drag reduction at either temperature or salinity are developed. These results are presented and discussed in detail.

Molecular characteristics of sodium desoxyribonucleate

Studies bt three different physical methods on a sample of Na desoxyribonucleate are described. 1. 1. Light-scattering studies indicate that the molecular length decreased from 6800 A for solvent ionic strength zero, to 4500 A for ionic strength 0.02. The molecular weight was found to be 4.5·10 6. 2. 2. Viscosities extrapolated to zero shear rate indicate an axial ratio of 330. 3. 3. Electron microscopic photographs reveal filamentous particles whose diameters are in agreement with values obtained by indirect methods.