Inactivation of lipid-enveloped viruses in proteins by caprylate.

The use of caprylate for the inactivation of lipid-enveloped viruses in biologically active proteins both plasma derived and produced by cell culture was evaluated. Viruses consisted of herpes simplex virus type I, vesicular stomatitis virus, vaccinia virus, and Sindbis virus. Utilizing the dissociation reaction and varying the concentration of the ionized form of caprylate, a specific amount of the nonionized form of caprylate was maintained over a wide pH range. Virus-spiked protein solutions contacted with caprylate provide rapid virus inactivation under a variety of conditions while maintaining the integrity of the respective protein or activity. With the exception of coagulation factor AHF, protein and biological activity yield were essentially quantitative. Caprylate is removed after treatment by size exclusion chromatography or anion/cation exchange adsorption of the protein, followed by buffer wash.

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.

Localization of the substrate and oxalacetate binding site of succinate dehydrogenase.

Succinate dehydrogenase is composed of two subunits, one of molecular weight 70,000, containing FAD in covalent linkage to a histidyl residue of the polypeptide chain, the other subunit of molecular weight 30,000. The fact that substrate, substrate analogs, and oxalacetate prevent inactivation of the enzyme by thiol-specific agents indicates that a thiol group must be present in close proximity to the flavin. Comparison of the incorporation of radioactivity into each subunit in the presence and absence of succinate or malonate shows that both substrate and competitive inhibitors protect a sulfhydryl group of the 70,000-molecular weight subunit. This indicates that a thiol group of the flavoprotein subunit is part of the active site. Similar investigations using oxalacetate as a protecting agent indicate that the tight binding of oxalacetate to the deactivated enzyme also occurs in the flavoprotein subunit, and may involve the same thiol group which is protected by succinate from alkylation by N-ethylmaleimide. It is clear, therefore, that not only the flavin site but also an essential thiol residue are located in the 70,000-molecular weight subunit. A second thiol group, located in the 30,000-molecular weight subunit, also binds N-ethylmaleimide covalently under similar conditions, without being part of the active site. Succinate, malonate, and oxalacetate do not influence the binding of this inhibitor to the thiol group of the lower molecular weight subunit. Using maleimide derivatives of nitroxide-type spin labels, it has been possible to demonstrate the presence of two types of thiol groups in the enzyme which form covalent derivatives with the spin probe. When the enzyme is treated with an equimolar quantity of the spin probe, a largely isotropic electron spin resonance spectrum is obtained, indicating a high probe mobility. When this site is first blocked by treating the enzyme with an equimolar quantity of N-ethylmaleimide, followed by an equimolar amount of spin label, the label is strongly immobilized with a splitting of 64 gauss. It is suggested that the sulfhydryl group which is involved in the immobilized species is at the active site.