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The preparation of affinity columns that contain insulin attached to Sepharose in a targeted manner by way of biotin-avidin noncovalent bonds is described. Insulin was acylated selectively at the amino terminus of the B chain with the N-hydroxysuccinimido ester of biotin to form NaB'-biotinylinsulin. The ability of this modified insulin to stimulate rat epididymal adipocytes was (mean i SD) 94 + 9.6% (PF 0.05) that of the control insulin. NaOB'-Biotinylinsulin displaced 4-hydroxyazobenzene-2'-carboxylic acid from avidin, demonstrating affinity for this protein. The formation of the Na2B'-biotinylinsulin-avidin complex was visualized by cellulose acetate electrophoresis at pH 4. Na6B'-Biotinylinsulin combined with avidin attached to Sepharose to form affinity columns in which the hormone was attached to the support by strong noncovalent -bonds. The determination of the loading of avidin-Sepharose columns with biotinylinsulin was greatly facilitated by the attached biotin which provided a marker whose concentration could be assessed accurately by titration with avidin. Biotinylinsulin attached to avidin-Sepharose beads retained the ability to stimulate rat epididymal adipocytes. The activity of several samples of these beads was about 15% that of free biotinylinsulin, based on the amount of biotinylinsulin anchored to the support. The advantages of biotinylated hormones for the targeted attachment of hormones to solid supports are discussed. The effectiveness of affinity columns for receptor studies is dependent on the highly specific complementarity between hormone and receptor, and consequently the binding region of the hormone should not be modified. The problem of attaching polyfunctional molecules such as insulin to solid supports without disturbing their receptor-binding potential represents a challenging problem which we address in this communication.Insulin was selectively acylated with biotin ( Fig. 1 upper) at the amino terminus of the B chain to form NaB -biotinylinsulin (Fig. 1 lower). This compound, named "biotinylinsulin" for simplicity's sake, was prepared with the expectation that it would exhibit affinity for insulin receptors and also toward the egg-white protein, avidin (for a review, see ref. 1). As a result of this bivalency, it provides the basis for the targeted preparation of selective insulin receptor affinity columns. Such columns result when the biotinylinsulin is added to a solid support containing covalently attached avidin. Fundamentally they are based on the high affinity of biotin for avidin (dissociation constant, KD, approximately 1015).The advantages of the method are: (i) biotin can be attached to the hormone in a targeted manner; (ii) the chemical manipulations are performed with the free hormone, and thus their effect on binding capacity and biological activity can be readily assessed by bioassay; (iii) the attachment of the modified hormone to the support involves a strong noncovalent bond that forms specifically and spontaneously on simple mixing of the Schleicher an...

The purification of human fibroblast interferon by chromatography on Blue Sepharose and high-performance liquid chromatography is described. The amino acid composition and a partial sequence of the homogeneous protein are reported. The NH2 terminus was determined to be NH2-Met- Ser-Tyr-Asn-Leu-Leu-Gly-Phe-Leu-Gln-Arg-Ser-Ser-Asn-PheGln-X-Gln-Lys.Other laboratories have reported on the purification and partial structural analysis of human fibroblast interferon (1, 2). We present a novel purification and some additional sequences of human fibroblast interferon. Interferon used in our studies was prepared in serum-free medium and was purified by a procedure based on the combination of affinity chromatography and high-performance liquid chromatography (HPLC).EXPERIMENTAL PROCEDURES Interferon Production and Assay. Crude fibroblast interferon was produced as described by Havell and Vilcek (3), except that serum was omitted from the overnight induction medium. Interferon titers were determined by a cytopathic effect inhibition assay that was modified so that the entire assay could be performed within 16 hr (4). All interferon titers were expressed in terms of reference units/ml, calibrated against the reference standard for human leukocyte interferon (G-023-901-527) provided by the Antiviral Substances Program of the National Institute of Allergy and Infectious Diseases, Bethesda, MD.Purification of Human Fibroblast Interferon on Blue Sepharose. Sodium chloride was added to medium containing interferon to a final concentration of 1 M, and the solution was then pumped onto a 25-ml Blue Sepharose CL-6B (Pharmacia) column (1, 5) at room temperature at a rate of 2.5 ml/min. The unfractionated interferon was maintained on ice during the loading process. The column was washed with 250 ml of sodium phosphate buffer (50 mM Na2HPO4 adjusted to pH 7.2 with HCI) containing 1 M NaCl and 30% (vol/vol) ethylene glycol. The interferon was eluted with the same solution containing 50% (vol/vol) ethylene glycol. Peak fractions of activity were pooled and stored at 40C until used (Fig. 1). Activity appeared to be stable for at least 3 mo at 4VC or in liquid nitrogen.In preparations having a low initial titer, a second passage through Blue Sepharose was required. In these instances, when a total of 25 liters of culture medium containing interferon had been chromatographed, the peak fractions from five columns were pooled and adjusted to 10% (vol/vol) ethylene glycol, 2 M NaCl, and 50 mM Na2HPO4 (pH 7.2). This material was then applied to another Blue Sepharose column. The column was then washed with 250 ml of the sodium phosphate buffer containing 2 M NaCl and 30% ethylene glycol. Some interferon was eluted at this step. The remaining interferon was eluted with the same solution containing 50% ethylene glycol. Interferon that eluted from the second Blue Sepharose column with both 30% and 50% ethylene glycol was satisfactory for use in the next step of the purification. The specific activity of the interferon that was deemed sati...

Percutaneous transluminal coronary angioplasty is often complicated by thrombotic abrupt vessel closure in patients with unstable angina pectoris. The present multicentre trial was performed to determine the feasibility of two-dose regimens of recombinant hirudin (r-hirudin) compared to standard heparin in patients undergoing coronary angioplasty for unstable angina, and to investigate the effects of the different treatment regimen on markers of coagulation activation. At five participating centres, 61 patients were randomly enrolled in one of two sequential groups of r-hirudin (group 1: 0.3 mg.kg-1 i.v. bolus, 0.12 mg.kg-1.h-1 i.v. infusion; 21 patients; group 2: 0.5 mg.kg-1 i.v. bolus, 0.24 mg.kg-1.h-1 i.v. infusion; 19 patients) or in a heparin control group (150 IU.kg-1 i.v. bolus, 20 IU.kg-1.h-1 i.v. infusion; 21 patients). Antithrombotic therapy was started immediately before coronary angioplasty and continued for 24 h. This was followed by a low-dose anticoagulant infusion for another 24 h (r-hirudin: 0.04 mg . kg-1 . h-1; heparin: 7 IU . kg-1 . h-1). Activated partial thromboplastin time, r-hirudin plasma concentrations by both immunological and functional assay, thrombin-hirudin complex, thrombin-antithrombin III complex, soluble fibrin, and prothrombin fragment 1 + 2 were closely monitored. The median partial thromboplastin time prolongations at 24 h vs baseline were found to be 1.9-fold and 2.3-fold in r-hirudin group 1 and dose group 2, respectively, and 3.0-fold in the heparin group. There was a dose-dependent correlation between partial thromboplastin time and the r-hirudin plasma levels (r = 0.61). In five of 21 patients of dose group 1, three of 19 patients of dose group 2, and 10/21 patients of the heparin group, partial thromboplastin time values exceeding the predefined target range prompted an interruption of the infusion. One major bleeding complication occurred in dose group 2. The functional assay for the estimation of r-hirudin plasma concentrations showed excellent correlations to the immunological technique (r = 0.99). Differences between the thrombin-hirudin complex levels could not be observed. Increased concentrations of thrombin-antithrombin III complex, soluble fibrin, and prothrombin fragment 1 + 2 were seen 4-8 h after coronary angioplasty and after reduction of the high-dose therapy in dose group 1 when compared with dose group 2 and the heparin group, respectively. Based on coagulation tests the present study showed the feasibility of a periprocedural antithrombotic regimen with r-hirudin for patients undergoing coronary angioplasty for unstable angina. In addition to the partial thromboplastin time the determination of r-hirudin plasma levels by a chromogenic substrate assay considerably improves the monitoring of therapy. The lower dose r-hirudin regimen seems to be suboptimal as periprocedural anticoagulation in coronary angioplasty patients as indicated by markers of thrombin generation and thrombin activity.