Rapid purification of staphylococcal enterotoxin B by high-pressure liquid chromatography.

The Staphylococcus aureus enterotoxins represent a group of proteins that cause emesis and diarrhea in humans and other primates. We have developed a rapid two-step high-pressure liquid chromatography (HPLC) procedure for purification of staphylococcal enterotoxin B (SEB). Sterile filtrates (2.5 liters) of strain 10-275 were adsorbed directly onto a reversed-phase column (50 mm by 30 cm Delta Pak; 300 A [30 nm], 15 microns, C18). SEB was obtained by using a unique sequential gradient system. First, an aqueous ammonium acetate to acetonitrile gradient followed by an aqueous trifluoroacetic acid (TFA) wash was used to remove contaminants. A subsequent TFA to acetonitrile-TFA gradient eluted the bound SEB. Further purification was obtained by rechromatography on a cation-exchange column. From 35 to 45% of the SEB in starting filtrates was recovered. Analysis by immunoblotting of samples separated on sodium dodecyl sulfate-polyacrylamide gels indicated that HPLC-purified SEB exhibited immunological and biochemical properties similar to those of the SEB standard. Induction of an emetic response in rhesus monkeys showed that the HPLC-purified toxin also retained biological activity.

Rapid isolation of thymosin beta 4 from thymosin fraction 5 by preparative high-performance liquid chromatography.

We have developed a rapid, efficient, and reproducible two-step method for the purification of thymosin beta 4 (T beta 4) from thymosin fraction 5 (TF5). This purification is based on the use of high-performance preparative/semi-preparative and analytical reversed-phase (Delta-Pak C18) chromatographic columns. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid compositional analysis have shown that natural, purified T beta 4 is identical to synthetic T beta 4. This procedure can be used to isolate other biologically active peptides from TF5 in sufficient quantity for characterization.

Scale-up methodology for the preparative purification of peptide M.

An octadecapeptide, peptide M, the epitope of a retinal protein that induces experimental autoimmune uveitis, was synthesized and purified by preparative reversed-phase chromatography. The flow-rate and gradient conditions for maximum separation of impurities were determined on a 30 x 0.39 cm I.D. column of Delta Pak (15-microns spherical C18-bonded silica with 300-A pores). The maximum amount of peptide that was resolved under these conditions was then determined experimentally. Using a scale factor dependent on the square of the column diameters, the flow-rate and amount loaded were increased 164 times on a 30 x 5 cm I.D. column of the same packing. The same resolution was achieved. Batches of 200-342 mg were chromatographed with reproducible results, providing a total yield of 394 mg of pure peptide.

Immunochemical and structural similarities in toxin A and toxin B of Clostridium difficile shown by binding to monoclonal antibodies.

Clostridium difficile toxins A and B were shown to share immunochemical and structural features, including shared sequential epitopes. Nineteen hybridomas generated after immunization of mice with a mixture of toxoids produced monoclonal antibodies, all IgM(x), which bound to toxin A and toxin B in a solid-phase radioimmunoassay (RIA). None of the antibodies neutralized the cytotoxicity of either toxin, alone or in pairs, nor did they neutralize mouse lethality. The antibodies did not inhibit hemagglutination by toxin A, and none of those tested neutralized the toxin's enterotoxic activity. Studies of binding of antibodies to native toxins in the RIA showed that the antibodies differed in their recognition of the toxins. Many of the antibodies bound with higher avidity to toxin A than to toxin B. In Western blots, all the antibodies recognized both toxins in the native state; in addition, some antibodies recognized the minor cytotoxic species of toxin B. When the toxins were denatured and reduced, five antibodies bound to both toxins, five to A only, and nine to neither, demonstrating that the antibodies had different epitope specificities. Further structural comparisons were made by investigation of mol. wts, subunit structures and amino acid compositions. The native mol. wts of toxin A and toxin B, as determined by electrophoresis to equilibrium in 4-30% polyacrylamide gel electrophoresis (PAGE), were 430,000 and 368,000, respectively. Denatured and reduced toxins each had a single subunit of 315,000. Both toxins had about 50% hydrophobic amino acids.

Hereditary warfarin resistance. Investigation of a rare phenomenon.

A 57-year-old black woman required a daily dosage of 50 mg of warfarin sodium to maintain her prothrombin time in a therapeutic range. The central volume of distribution and clearance of warfarin were normal for this patient. These findings, combined with the patient's requirement for plasma warfarin levels four times greater than those usually required to achieve adequate anticoagulation, indicated that the relative resistance was due to altered pharmacodynamics of warfarin. The only child of the propositus, a daughter, showed a similar relative resistance, confirming that this family is the third to be reported with hereditary resistance to warfarin.

Femtomolar ion-pair high-performance liquid chromatographic method for determining Dns-polyamine derivatives of red blood cell extracts utilizing an automated polyamine analyzer.

An ultra-sensitive automated method for the determination of polyamines in red blood cell extracts by ion-pair reversed-phase high-performance liquid chromatography is described. The 5-dimethylaminonaphthalene-1-sulfonyl derivatives of putrescine, 1,6-diaminohexane, spermidine, and spermine are separated on a muBondapak C18 column using 1-heptanesulfonic acid and acetonitrile as the mobile phase. All compounds are eluted within 28 min subsequent to the initial injection. The method has a lower detection limit of 25 fmoles on column. Because of the simplicity and ease of operation, the method is applicable for use in either the research or clinical laboratory.