Thomas F. Busby

Separation of Macromolecules by Ultrafiltration: Influence of Protein Adsorption, Protein-Protein Interactions, and Concentration Polarization

Ultrafiltration through microporous membranes is a well-established technique for concentrating dilute protein solutions and separating proteins from low molecular weight solutes such as salts or ethanol (Friedli et al., 1977; Guthorlein, 1977; Mercer, 1977), or from much larger particles such as cells (Colton et al., 1975). However, the use of this tool for fractionating proteins according to size has progressed less rapidly. Several difficulties can be identified: non-uniform pore size protein adsorption concentration polarization protein-protein interactions

Thermal stability and ligand-binding properties of human plasma α1-acid glycoprotein (orosomucoid) as determined with fluorescent probes☆

The fluorescence of 1,8-anilinonaphthalene sulfonate is enhanced and blue-shifted upon binding to alpha 1-acid glycoprotein, a human plasma protein of uncertain function. Fluorescence titrations of delipidated protein indicate at least two classes of binding sites having dissociation constants of 0.33 microM and 12 microM at 25 degrees C in 0.02 M potassium phosphate/0.15 M NaCl, pH 7.4. Exclusion chromatography measurements indicate only 1 binding site per mol protein, suggesting that the heterogeneity is due to differences between protein molecules, the origin of which remains unclear. The fluorescence of a mixture of dye and protein is progressively diminished upon addition of ethanol and other organic solvents whose presence could be detected at concentrations as low as 100 mM. Addition of the adrenergic drug propranolol to a mixture of alpha 1-acid glycoprotein (2.5 microM) and 1,8-anilinonaphthalene sulfonate (4 microM) caused a hyperbolic decrease in dye fluorescence to 30% of the initial value, with half-maximal response near 1 microM propranolol. When the protein-dye mixture was heated, the fluorescence of the dye exhibited a reversible downward transition with midpoint near 65 degrees C, compared to a midpoint of 58.5 degrees C obtained by intrinsic fluorescence in the absence of dye. This stabilization was confirmed with fluorescein-labeled protein, whose fluorescence polarization revealed a melting transition at 58.8 degrees C in the absence of ligands which increased by 5-6 Cdeg in the presence of 1,8-anilinonaphthalene sulfonate or propranolol. The sensitivity of 1,8-anilinonaphthalene sulfonate fluorescence to changes in the conformation and ligand environment of alpha 1-acid glycoprotein should facilitate efforts to understand the structure and function of this acute-phase reactant.

Evaluation of a new LightCycler reverse transcription-polymerase chain reaction infectivity assay for detection of human parvovirus B19 in dry-heat inactivation studies.

BACKGROUND: Human parvovirus B19 (B19) is a widely distributed infectious agent, which causes a variety of illnesses including erythema infectiosum (fifth disease) especially in children, arthritis, aplastic crisis, and hydrops fetalis. B19 can be transmitted from asymptomatic blood donors to recipients of their blood components. Fifth disease has been reported in patients receiving red blood cells, platelets, solvent/detergent‐treated plasma, and clotting factor concentrates.

Removal of Polyethylene Glycol from Proteins by Salt‐Induced Phase Separation

Abstract. 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.

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°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.

Characterization of Protein-Protein and Protein-Ligand Interactions by High Performance Size Exclusion Chromatography

Abstract HPLC has been used in our laboratory to characterize a wide range of protein-protein and protein-ligand interactions. In a study of the dissociation and recombination of human chorionic gonadotropin subunits, HPLC provided a fast and sensitive method for directly observing the state of association of samples equilibrated under various conditions. The α subunit (15 Kd) was easily resolved from the β subunit (23 Kd) using a Toyo Soda type SW 3000 column (0.8 × 60 cm) eluting at 1 ml/min. The subunit was poorly resolved from the intact hormone (38 Kd) in agreement with results obtained using conventional exclusion media. In another study, the same column was used to assess the degree of aggregation of various protease inhibitors (antithrombin III (AT III), C1-inactivator and α1-proteinase inhibitor) after heating, as part of an effort to determine conditions under which these potentially therapeutic proteins might withstand pasteurization to reduce the risk of transfusion hepatitis. The ability of A...

Establishment of a porcine parvovirus (PPV) DNA standard and evaluation of a new LightCycler nested-PCR assay for detection of PPV

Porcine parvovirus (PPV) is a major causative agent in a syndrome of reproductive failure in swine. In validation (viral clearance) studies, PPV is a model of non-enveloped viruses and is widely used instead of human parvovirus B19, which causes a variety of illnesses including erythema infectiosum (fifth disease) in children and hydrops fetalis in pregnant women. To improve the sensitivity of current PCR-based assays for detection of PPV and to standardize the quantification of PPV, we have developed a lightcycler (LC) nested-PCR (nPCR) assay and constructed a PPV DNA standard evaluated in the LC nPCR assay. The PPV DNA standard, a plasmid termed pPPV, encodes a 3.3 kb PPV NADL-2 genome fragment. One genome copy equivalent (gce) of PPV equals 6.7 attograms of pPPV. The LC nPCR assay is a simple and specific method developed for detection of PPV strains but not any other viruses including members of Parvoviridae. The first 25-cycle PCR with outer primers chose by comparative analysis of 12 primers in 21 different combinations and a second 45-cycle PCR with inner primers amplify 286 and 251 bp fragments of PPV genome, respectively, for 40 min with a sensitivity of approximately 100 gce per assay (ml). By using the LC nPCR assay for analysis of PPV samples with known infectivity, we found that one 50% tissue culture infectious dose (TCID(50)) equals 1.93 +/- 0.24 log(10) gce.

Dynamic equilibria between subcomponents of CĪ, the first component of human complement

C1r and C1s, the serine protease components of activated C1, form a tetramer in the presence of Ca2+. The stability of this tetramer is sufficient that its association with the third component, C1q, has been successfully treated as a reversible bimolecular equilibrium reaction [Siegel and Schumaker, Molec. Immun. 20, 53-66 (1983)]. We have used the fluorescence anisotropy (A) of fluorescein-labeled C1s (s*) to monitor assembly and subcomponent exchange in 0.15 mol/l NaCl, 0.001 mol/l Ca2+ 0.02 mol/l Tris, pH 7.4. Addition of q to r2s*2 causes a small but measurable delta A of 0.01-0.02. The response is too fast to measure at 37 degrees but can be readily followed at 4 degrees where t 1/2 = 0.6 min when [q] = [r2s*2] = 0.5 mumol/l. The increase in A can be readily reversed by dilution or by addition of unlabeled C1s. Slow incremental addition of q to a solution of r2s*2 produces a dose-dependent delta A from which stoichiometry and dissociation constants can be derived. Measurements of Kd as a function of temperature establish an inverse temperature dependence with delta H = -15 kcal/mol and a value of Kd = 0.031 mumol/l at 37 degrees (delta G = + 11, T delta S = -26 kcal/mol). Thus, the assembly process appears to be entropy-driven presumably due to the exclusion of structured water from protein-protein interfaces in the complex.

Heat stability of lyophilized C1 inactivator concentrates

Abstract. Heat stability of lyophilized C1 inactivator (C1‐INA) concentrates of intermediate and high purity has been investigated under several heat treatment protocols that include heating for 96 and 192 h at 68°C and for 10 h at 80, 90 and 100°C. Both types of concentrate showed high stability in functional activity, with not more than 5% loss in any of the time‐temperature combinations evaluated. However, the C1‐INA antigen from both concentrates showed small but progressive changes in crossed immunoelectrophoretic pattern, in proportion to the intensity of heat treatment. High‐pressure size‐exclusion chromatography revealed only minimal signs of aggregation in the high‐purity concentrate, but a significant and progressive aggregation of nonspecific protein contaminants present in the intermediate‐purity concentrate, making the high‐purity concentrate preferable for heat treatment.

Biological and Physical Properties of Fibronectin Pasteurized in the Presence of Stabilizers

Abstract. Interest in human plasma fibronectin (Fn) as a potential clinical product for replacement therapy in septic patients has prompted the search for stabilizers to protect the protein from heat denaturation during pasteurization designed to inactivate hepatitis viruses. Fn was pasteurized (60°C, 10 h) in the presence of either citrate, tricarballylate, sucrose or four mixtures of lysine, glucarate, gluconate or citrate which had been found to increase the denaturation temperature of Fn by 19°C. All but a citrate/gluconate mixture were effective in preventing aggregation as measured by dye fluorescence, light scattering, gel filtration and electrophoresis. Binding to gelatin was retained and immunological activity was only slightly diminished compared to a sample heated without stabilizers. Opsonic activity was measured as ability to mediate the uptake of 125I‐gelatin‐coated polystyrene beads by attached human monocytes. Fn heated without stabilizers underwent a transient increase in activity which was traced to formation of aggregates having elevated specific activities. Pasteurized samples had slightly elevated opsonic activities with no detectable aggregates present, while the unstabilized control was inactive. These results indicate that the physical properties of Fn as well as the functional activities of the gelatin‐ and cell‐binding domains can be protected against thermal denaturation by various compounds.