In vitro characterization of antithrombin III concentrates--a single-blind study.

Twenty-three lots of five antithrombin III (AT III) concentrates from four manufacturers were analyzed in a single-blind study. All the preparations had been virus-inactivated by pasteurization, and one concentrate had also been treated with solvent/detergent (S/D). AT III activities were determined using two thrombin-based and one factor Xa-based chromogenic substrate assays. AT III antigen was measured by kinetic nephelometry. All AT III assays were tested against the first international reference preparation coded 72/1. In addition, AT III was characterized by crossed immunoelectrophoresis in the presence of heparin and by gel filtration. The following were quantified: heparin cofactor II activity and antigen content, heparin activity, thrombin-AT III complexes, AT III-protease complexes, total protein, albumin, immunoglobulins, glucose and pH. The AT III concentrates differed markedly in terms of their purity and potency. The specific activities of AT III and the ratios of AT III activity to antigen content ranged from 3.4 to 6.9 and from 0.63 to 0.84, respectively. The highest values were found in five lots of the concentrate that had been treated by both pasteurization and S/D. This preparation was the only one that was virtually free of denaturated AT III, as judged by crossed immunoelectrophoresis. Marked batch-to-batch variation in AT III potencies was found in two out of the five preparations analyzed. In two out of five lots from one manufacturer, the measured potencies were more than 10% lower than the declared potencies.(ABSTRACT TRUNCATED AT 250 WORDS)

High-performance liquid chromatography and diode-array detection for the identification of peptides containing aromatic amino acids in studies of endorphin-degrading activity in human cerebrospinal fluid.

Diode-array UV detection has been adapted for analysis of opioid peptides and their metabolic fragments differing in aromatic amino acid content. In combination with high-performance liquid chromatography, the technique allowed a direct and rapid discrimination between peptides containing phenylalanine, tryptophan and tyrosine, or a combination of these residues. Enkephalin fragments with either tyrosine or phenylalanine, or both, were identified after digestion of the pentapeptide with proteolytic activity recovered from human cerebrospinal fluid. The N-terminal tyrosine-containing fragment of dynorphin A was identified after hydrolysis of the peptide by a cerebrospinal fluid endopeptidase. The study was extended to the analysis of some non-opioid peptides. The Tyr1 analogue of delta-sleep-inducing peptide was easily distinguished from the authentic compound with a tryptophan at the N-terminus. Results indicated that the technique was useful for discriminating between dipeptides differing in aromatic residues that were unresolved by high-performance liquid chromatography.

Ion-exchange high-performance liquid-chromatography steps in peptide purifications.

Ion-exchange high-performance liquid chromatography (HPLC; on Ultropac TSK DEAE and CM) is compared with conventional soft-gel ion-exchange chromatography in identical peptide purifications. The results show that separating properties are similar, but as expected, ion-exchange HPLC has a much higher resolving capacity and a higher sensitivity, and allows a considerably shorter total separation time. The same buffer systems as for conventional ion-exchange chromatography can be used, including urea to solubilize large peptides, if care is taken not to exceed the pH limits set by the column matrix. A complete purification scheme by HPLC in the nanomolar range, utilizing exclusion, ion-exchange, and reverse-phase chromatographies, is given with a complex peptide mixture from a digest of a large protein. Similar steps as in conventional soft-gel schemes can be utilized. It is concluded that ion-exchange HPLC is a suitable complement to commonly used reverse-phase HPLC steps and that it permits high speed and sensitivity over wide ranges of peptide sizes and amounts.

Analytical isotachophoresis: a new method for analysis of cerebrospinal fluid proteins. A preliminary report.

Isotachophoresis of CSF proteins seems to be a very promising method. Very small CSF samples, a few mul of concentrated and 15-30 mul of unconcentrated CSF, can be quickly analysed (30-60 min), and the results immediately obtained on a recorder. Using unconcentrated CSF, losses due to concentration procedures, are avoided. Low-molecular weight compounds, e.g. in CSF ultrafiltrates, can also be examined. The method gives high resolution, is reproducible, and is easy to perform. The isotachophoretic findings have been compared with those of electrofocusing.