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Biomedical subjects

K C Ingham

Publications and source records attributed to K C Ingham.

At least 73 records · Page 4Linked to original sources
PubMed1984

Pasteurization of C1 inactivator in the presence of citrate salts.

Conditions have been determined under which the C1 inactivator (C1-INA) can be pasteurized to reduce the risk of transfusion hepatitis associated with its use for replacement therapy in patients with genetic or acquired deficiencies. Recovery of 90% of the biological and immunological activity of a C1-INA concentrate was achieved following heat treatment for 10 h at 60 degrees C in the presence of 3 M potassium citrate. Crossed immunoelectrophoresis in heparinized agarose was used to demonstrate the ability of the pasteurized C1-INA to bind heparin and to form a precipitation pattern with antibody which was almost indistinguishable from that of an unheated control. High pressure liquid chromatography and enhancement of the fluorescence of 1,8-anilinonaphthalene sulfonate were used to show that other proteins present in the concentrate were also stabilized.

Citrates↗
PubMed1983

Fluid-phase interaction between human plasma fibronectin and gelatin determined by fluorescence polarization assay.

Previous studies of the binding properties of fibronectin (Fn) have utilized methods whereby one or the other macromolecule was immobilized on a solid phase. In order to examine the interaction between human plasma Fn and gelatin in solution, the latter was labeled with fluorescein isothiocyanate (FITC) whose fluorescence polarization (P) served as a sensitive indicator of the formation of soluble complexes. Changes in P were detectable at Fn concentrations below 10(-9) M and continued up to concentrations above 10(-6) M at pH 7.3 and 25 degrees C. Fractionation of FITC-gelatin by exclusion chromatography and titration of selected fractions revealed a trend towards higher affinity with increasing size. A high-molecular-weight fraction comprised of beta and gamma components and a low-molecular-weight fraction comprised primarily of alpha chains exhibited sigmoidal increases in P (apparent positive cooperativity) with 50% saturation near 10(-9) and 10(-8) M Fn, respectively. By contrast, a 42-kDa chymotrypsin-generated Fn fragment which retains the ability to adhere to gelatin-Sepharose exhibited normal (noncooperative) binding to both gelatin fractions with Kd = 7 X 10(-7) M. In all cases, the increase in P could be reversed by addition of excess unlabeled gelatin or urea. The interaction of FN with FITC-gelatin provides the basis for a fast and sensitive determination of Fn levels in plasma and other fluids. Interference caused by other proteins such as albumin, which has an affinity for the fluorescein moiety, could be minimized by addition of 1.0 M NaCl which had no effect on the interaction between Fn and gelatin.

Chromatography, Affinity↗
PubMed1983

Interaction of plasma fibronectin with gelatin and complement C1q.

A variety of techniques have been used to examine the interaction of human plasma fibronectin (Fn) with complement C1q in comparison to that with gelatin in phosphate buffered saline at pH 7.4. The precipitation of 3H-Fn by polyethylene glycol (PEG) was shifted to much lower concentrations of the polymer by addition of gelatin, and to a lesser extent, by C1q. Precipitation of 3H-Fn in the presence of C1q was close to that of C1q alone under identical conditions suggesting an affinity of Fn for solid phase C1q; a similar interaction was seen with heat-insolubilized C1q. Fibronectin bound tightly to gelatin-Sepharose and C1q-Sepharose and this binding could be inhibited by gelatin but not by C1q. The presence of gelatin retarded the anodal migration of Fn during immunoelectrophoresis under physiological conditions whereas C1q had an effect only at low ionic strength. Exclusion chromatography of Fn, alone and preincubated with gelatin or C1q, was also consistent with the formation of strong complexes with gelatin but not with C1q, whereas similar mixtures of Fn and gelatin exhibited a fast-sedimenting boundary and marked depletion of the 12S Fn peak. Titration of fluorescein-labeled alpha 2 chains of type I collagen with Fn produced an increase in fluorescence polarization which could be reversed by addition of unlabeled alpha 2 chains or gelatin but not by C1q or the pepsin-derived collagen-like domain of C1q. These observations indicate that the fluid-phase interaction of Fn with C1q is much weaker than that with gelatin but that Fn does have appreciable affinity for solid-phase C1q. Such interaction could signify a role for Fn in the clearance of immune complexes from circulation.

Animals↗
PubMed1983

Thermal denaturation of alpha 1-protease inhibitor. Stabilization by neutral salts and sugars.

Alpha 1-protease inhibitor (alpha 1 PI), also called alpha 1-antitrypsin, may be useful for replacement therapy in a number of chronic or acute disorders. The risk associated with the possible presence of hepatitis virus can be greatly reduced by pasteurization at 60 degrees C for 10 h. A series of thermal denaturation profiles was obtained in the presence of various protein stabilizers using the increase in 1,8-anilinonaphthalene sulfonate fluorescence that accompanies protein denaturation. A parallel series of experiments was conducted to evaluate each additive for its capacity to protect the biological activity of alpha 1 PI. As much as 92% of the inhibitory activity against elastase and trypsin could be recovered after pasteurization in buffer containing citrate (1.2 M) and either EDTA (0.5 M) or gluconate (1.2 M). Loss of activity was not affected by protein concentration. In conclusion, conditions have been developed to protect the bulk of alpha 1 PI from denaturation during pasteurization, and this should give an added impetus to efforts to test the efficacy of this protein in various clinical conditions.

Anilino Naphthalenesulfonates↗
PubMed1982

Rapid methods for isolation of human plasma fibronectin.

Simplified procedures have been developed for isolation of human plasma fibronectin by affinity chromatography on gelatin-agarose. In one method, fibronectin is eluted with 3 M urea, and this reagent is quickly removed by adsorbing the protein onto heparin-agarose, followed by 0.4 M NaCl elution. In a shorter process, fibronectin is eluted from gelatin-agarose simply by decreasing the buffer pH below 6. After lyophilization the purified protein can be readily dissolved in water. The fraction not adsorbed to gelatin can be used to purify other proteins, including factor VIII whose procoagulant activity is quantitatively recovered.

Chemical Precipitation↗
PubMed1981

Mechanism of precipitation of proteins by polyethylene glycols. Analysis in terms of excluded volume.

The apparent solubilities of various proteins (14,000 to 670,000 daltons) were measured in the presence of polyethylene glycols (PEGs) of different sizes. All of the solubility curves, determined by measuring the protein concentration in the supernate of centrifuged mixtures, exhibited the characteristic linear dependence of log S (g/liter) on PEG concentration (%, w/v). For human albumin in PEG-4000 at pH 4.5, this linearity extended over a 1,000-fold range of solubility, even though the appearance of the sedimented phase changed from a viscous fluid to a white amorphous solid. The slope, beta, decreased from 0.27 to 0.09 with decreasing Mr of PEG from 20,000 to 400, but was insensitive to changes in solution conditions (pH, T, salts), suggesting the absence of specific chemical interactions between protein and polymer. This conclusion was supported by the observation that concentrations of PEG up to 30% (w/v) had no significant effect on the melting temperature of ribonuclease A. Furthermore, equilibrium dialysis measurements, as well as various spectral measurements, provided no evidence for such interactions. Using a steric exclusion model (Edmond, E., and Ogston, A. G. (1968) Biochem. J. 109, 569-576) and assuming that the chemical potential of the solid phase is constant, beta can be related to interaction coefficients calculated from co-volumes using the equivalent sphere radii of PEG (r2) and protein (r3). Although good agreement was obtained for albumin in PEG-20,000, the predicted dependence of beta on r3 was greater than observed and the predicted dependence of beta on r2 was of opposite direction to that observed. However, the interaction coefficient determined from the equilibrium dialysis measurements of albumin and PEG-1000 agreed with the predicted value. Thus, the exclusion of low concentrations of PEG by albumin can be explained by a simple excluded volume model, whereas the exclusion of protein out of solution by PEG appears to be more complex.

Animals↗
PubMed1981

Thermal denaturation of antithrombin III. Stabilization by heparin and lyotropic anions.

Antithrombin III is of potential value for replacement therapy in patients with acquired or congenital deficiencies. Pasteurization of the purified inhibitor for 10 h at 60 degrees C can reduce the risk of transfusion hepatitis. Addition of appropriate stabilizers can largely prevent the loss of antithrombin activity which otherwise occurs during pasteurization. Studies of the mechanism of denaturation and stabilization have been facilitated by the use of 8-anilino-1-naphthalene sulfonate which binds weakly to the inhibitor and whose fluorescence undergoes a sigmoidal response to increasing temperature. The extent of the increase in 8-anilino-1-naphthalene sulfonate fluorescence correlates roughly with the loss of antithrombin activity and with the extent of protein aggregation as determined by high pressure liquid chromatography. The midpoint, Td, of the thermal denaturation curve increases by 13 degrees C and 19 degrees C in the presence of 0.5 M and 1.0 M sodium citrate, respectively. Phosphate, sulfate, and EDTA are also strong stabilizers while the chaotropic anions, iodide and thiocyanate are potent destabilizers. Heparin at 10 mg/ml increases Td by 7 degrees C, presumably through a direct binding mechanism; chondroitin sulfate and hyaluronic acid have no effect. Samples pasteurized for 10 h at 60 degrees C in the presence of 0.5 M and 1.0 M citrate retain essentially full activity but exhibit evidence of minor alterations in their interaction with heparin.

Anilino Naphthalenesulfonates↗
PubMed1981

Fluorescent labeling of the carbohydrate moieties of human chorionic gonadotropin and alpha 1-acid glycoprotein.

A method for covalent attachment of a fluorescent molecule to the carbohydrate moieties of glycoproteins is described. The glycoproteins were oxidized with periodate under mild conditions selective for sialic acid (Van Lenten, L. and Ashwell, G. (1971) J. Biol. Chem. 246, 1889--1894). The resulting aldehydes were condensed with either dansylhydrazine, dansylethylenediamine, or fluoresceinamine followed by reduction with NaCNBH3 and NaBH4. Conjugates prepared with dansylhydrazine were found to be insufficiently stable for spectroscopic analysis, whereas the primary amines produced stable conjugates whose fluorescence polarization (P) was constant for several hours at 37 degrees C. The degree of labeling correlated roughly with the sialic acid contents of the vaious glycoproteins. Very little covalent incorporation was observed with albumin (which is devoid of carbohydrate) or with asialo alpha 1-acid glycoprotein. Exclusion chromatography in the presence of a dissociating agent was sometimes required to remove significant amounts of noncovalently adsorbed dye. Fluorescent-labeled alpha subunits of human chorionic gonadotropin were shown to recombine normally with native beta subunits. However, the labeling procedure appeared to compromise the ability of the beta subunits to recombine. Electrophoretic analysis produced evidence of covalent cross-linking between subunits following periodate oxidation of the intact gonadotropin. The possibility that primary amine groups of the protein compete with added fluorescent amines for reaction with periodate-generated aldehydes is discussed.

Chemical Phenomena↗
PubMed1980

Separation of macromolecules by ultrafiltration: removal of poly(ethylene glycol) from human albumin.

Mixtures of albumin and poly(ethylene glycol) (PEG) were used to elucidate some of the factors which influence the separation of macromolecules by thin-channel ultrafiltration. Several membranes which readily passed PEG-4000 in the absence of protein were found to exhibit increased rejection of the synthetic polymer when albumin was added to the system. Based on a comparison of filtration flux and net sieving properties, the PM-30 membrane of Amicon was chosen for further characterization. The increased rejection of PEG-4000 in the presence of albumin was independent of albumin concentration between 1 and 100 mg/ml and persisted even after albumin was removed and the system flushed with water. Overnight incubation of the membrane with trypsin restored the original sieving properties, indicating that the 'permanent' effects were due to irreversible adsorption to the membrane. By measuring flux over a 10(6)-fold range of albumin concentration it was possible to resolve the effects of protein adsorption, a saturable process which occurs at low protein concentration (< 0.01 mg/ml), from the effects of concentration polarization which occur at high protein concentration (> 0.1 mg/ml). Only the former process has an effect on the net sieving properties in this system. In spite of the adverse effects of protein adsorption, it was still possible to obtain efficient removal of PEG-4000 from albumin. Exchange of approximately 5 vols. of solvent at room temperature resulted in a 10-fold reduction in the concentration of PEG in the sample, with no loss of albumin, and no formation of albumin dimers.

Adsorption↗
PubMed1980

Removal of polyethylene glycol from proteins by salt-induced phase separation.

The use of poly(ethylene glycol) in the purification of plasma and cellular protein is somewhat complicated by the difficulty of removing it from the protein product. The method presented here, quickly and efficiently removes over 95% of the PEG by the simple addition of salts to induce an aqueous two-phase separation with the PEG in the upper phase and greater than 90% of the protein in the lower phase.

Orosomucoid↗