Shirley Miekka

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.

Inactivation of viral and prion pathogens by γ‐irradiation under conditions that maintain the integrity of human albumin

Background and Objectives The administration of therapeutic plasma protein concentrates has been associated with the real risk of transmitting viral diseases and the theoretical risks of prion transmission. Our objective was to determine if γ‐irradiation can inactivate viral or prion infectivity without damaging a protein biotherapeutically.

Influence of self-association of proteins on their precipitation by poly(ethylene glycol)

Abstract The influence of self-association on the precipitation of proteins by poly(ethylene glycol) (PEG) was investigated using several model self-associating proteins. These include α-chymotrypsin and chymotrypsinogen, β-lactoglobulin A and glutamate dehydrogenase. With few exceptions, conditions which promote self-association also enhance precipitation by PEG, while conditions which inhibit self-association also inhibit precipitation by PEG. Chymotrypsin and chymotrypsinogen self-associate at pH 8.5 and low ionic strength and, under these conditions, they were precipitated by moderate concentrations of PEG (10–20%, w v ). Low concentrations of salts ( m ) reversed the self-association and prevented precipitation up to 25% PEG. By contrast, salt did not prevent the precipitation of covalently cross-linked oligomers of chymotrypsinogen. In acid (pH 4.5), the effects of salts on both self-association and precipitation of these proteins were opposite to the effects at pH 8.5. Different salts were generally found to have similar effects, but SO42− was particularly effective in enhancing precipitation in acid. At pH 3 and 8, β-lactoglobulin A is primarily a monomer and was not precipitated by 25% PEG at these pH values. Maximum precipitation occurred at pH 4.7, which is the reported optimum for self-association. Glutamate dehydrogenase associates to polymers at neutral pH and was precipitated by 15% PEG. The combined presence of NADH and GTP (1 m m ) prevented polymer formation and abolished precipitation. Toluene further enhanced polymerization in the absence of cofactors but did not enhance precipitation. However, PEG was found to strongly inhibit the polymerization induced by toluene. The results of these studies suggest that it may be possible to selectively enhance or inhibit the PEG precipitation of a self-associating protein through manipulation of its oligomeric state.

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.

Heat-induced fragmentation of human plasma fibronectin.

Human plasma fibronectin was found to undergo fragmentation during heat-denaturation, leading to artifacts in SDS-polyacrylamide gel electrophoretic analyses. Electrophoretic patterns of heated samples showed a progressive decrease in intact fibronectin chains (225 kDa) which coincided with the appearance of increasing amounts of numerous smaller components having molecular weights ranging from 10 000 to 200 000. The fragmentation was temperature-dependent, being undetectable after 2 h at 60 degrees C, but detectable after 30 min at 70 degrees C or as little as 2 min at 100 degrees C. After 2 h at 100 degrees C, the intact monomer was no longer visible. Neither mercaptoethanol nor SDS was required for fragmentation. Sterile filtration or pretreatment with inhibitors of proteolytic enzymes had no effect. Treatment with amines did not diminish the degradation, indicating that the process differs from heat-fragmentation of alpha 2-macroglobulin and complement proteins, which occurs at a reactive internal thiolester bond. Fibronectin fragmentation was highly pH-dependent, being markedly accelerated under acidic conditions, suggesting that autolytic cleavage of the peptide chain at acid-labile aspartyl bonds was responsible for this phenomenon.

Pulmonary Delivery of Human Protein C and Factor IX

Recent advances in structural elucidation of numerous natural peptides and proteins1, enhanced understanding of their role in several physiological processes, and the use of biotechnological techniques for their production have stimulated considerable interest in establishing peptides and proteins as therapeutic agents. A major problem, aside from proteolytic degradation and physical alteration of the protein molecule at the site of administration, is the slow rate of transport of macromolecules across membrane barriers into the systemic circulation.

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.