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A high-performance affinity chromatography support based on silica has been developed for the immobilization of proteins containing primary amino groups. A hydrophilic polymer covalently bound to the silica surface minimizes nonspecific protein binding to the support while preserving high binding capacity. The Schiff base reaction involved in the coupling of a ligand to the affinity medium is rapid, allows the use of mild conditions during the coupling process, and results in a very stable linkage. Reaction parameters were studied for protein coupling to the affinity support to determine optimum binding conditions and dynamic capacity as a function of protein size. The stability of the ligand-matrix bond was determined. The performance and reproducibility of the affinity support are demonstrated by its use in the analysis of nitrophenyl sugar derivatives, purification of glycoproteins, and isolation of anti-bovine immunoglobulin G developed in rabbit.

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Because of its high selectivity, affinity chromatography is a preferred tool in the downstream processing of high-value proteins and peptides of therapeutic interest. This review examines the affinity supports currently available, and investigates the performance characteristics and properties required of the support matrices for improved affinity-based supports for large-scale purification of biomolecules. Parameters for optimizing an affinity chromatographic process, and the advantages of affinity-based separation for scaled-up systems are highlighted.

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The chromatographic separation of four proteins, cytochrome c, alpha 1-acid glycoprotein, ovalbumin, and beta-lactoglobulin, was achieved on a 4.6 X 250-mm wide-pore polyethyleneimine (PEI)-silica gel column (5-micron particles, 330-A pore size) with essentially baseline resolution using a 20-min linear gradient from 0.025 M potassium phosphate, pH 6.80, to 0.50 M potassium phosphate, pH 6.80. The back pressure of this anion-exchange column was 1000 psi at a flow rate of 1.0 ml/min. Protein recoveries averaged over 95% and protein capacity exceeded 33 mg for a single protein. Isocratic elution (0.040 M potassium phosphate, pH 6.8; flow rate, 0.50 ml/min) of ovalbumin gave a column efficiency of 15,700 plates/m with a peak asymmetry factor of 1.27. Resolution of these same four proteins on a 4.6 X 50-mm PEI-silica gel column occurred within 2 min. Nucleoside monophosphates were separated on the short PEI-silica column within 1 min with 0.01 M potassium phosphate, pH 2.58, at a flow rate of 6 ml/min which generated a column back pressure of 2000 psi.

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Hepatocytes can be maintained in culture for periods of a few hours to many days. This review summarizes the metabolic characteristics of these cultures and describes their use in studying the regulation of plasma protein synthesis. Hormones selectively stimulate the synthesis of certain proteins. Cortisol stimulates the synthesis of fibrinogen and other acute-phase proteins; whereas, insulin stimulates albumin synthesis. In the latter case insulin increases the rate of a nuclear process. Mediators elaborated by leukocytes stimulate acute-phase protein synthesis in hepatocytes. Plasmin-generated fibrin peptides stimulate fibrinogen synthesis via a leukocytic mediator. Lipoprotein synthesis is stimulated by fatty acids and is inhibited by albumin and other macromolecules. These and other processes are susceptible to detailed analysis using sub-cellular fractions (mRNA, nuclei, transcription factors, etc.) isolated from hepatocytes. Studies on fetal or embryonic hepatocytes and hepatomas are yielding information on the regulation of secretory protein synthesis during development and following neoplastic transformation.

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A rapid, convenient immunologic assay technic for fibrinogen is reported. Basically a latex agglutination, the assay technic, called kinetic latex agglutinometry, quantitates the increase in light transmission that occurs in a stirred suspension of anti-fibrinogen-coated latex beads after the addition of a plasma or whole blood sample containing fibrinogen. In this system the time "delta t" required for a given transmission change to occur is inversely proportional in a log-log relationship to the quantity of fibrinogen in the plasma sample. The reaction is linear over a fibrinogen concentration of greater than 100-fold. The sensitivity can be adjusted over a wide range, and the assay can quantitate as little as 10 ng fibrinogen. The assay can be used with either plasma or whole blood. When compared to the thrombin clotting time method of Clauss, the correlation coefficient is 0.99 for the plasma assay and 0.95 for the whole blood assay. As a one-step assay employing stable reagents and requiring approximately two minutes per assay for normal plasma, the method is ideally suited for use in the clinical laboratory.

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A rapid method for measuring factor VIII antigen in plasma is presented, based upon optical measurement of the rate of agglutination of a stirred suspension of latex particles coated with anti-factor VIII immunoglobulin, when mixed with plasma containing factor VIII antigen. A log-log relationship exists between factor VIII concentration and the reciprocal of the time required for a fixed absorbance change. The assay is rapid, requiring less than 3 min for normal plasma, involves stable reagents, and can be performed with conventional instrumentation. Antigen values in normal plasma a well as plasma from patients with von Willebrand's disease (angiohemophilia) correlate well with values measured by electroimmunoassay or by radioimmunoassay.

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A system of preparation of rat hepatocytes with extended viability has been developed to study the role of hormones and other plasma components upon secretory protein synthesis. Hepatocytes maintained in minimal essential medium reduced the levels of all amino acids in the medium except the slowly catabolized amino acids leucine, isoleucine, and valine, which steadily increase as the result of catabolism of liver protein. Although the liver cells catabolize 10-15% of their own protein during a 20-h incubation, the cells continue to secrete protein in a linear fashion throughout the period. The effects of insulin, cortisol, and epinephrine on general protein synthesis, and specifically on fibrinogen and albumin synthesis, have been tested on cells from both normal rats and adrenalectomized rats. Cells from normal animals show preinduction of tyrosine amino transferase (TAT), having at the time of isolation a high level of enzyme which shows only an increase of approximately 60% upon incubation with cortisol. In contrast, cells from adrenalectomized animals initially have a low level of enzyme which increases fourfold over a period of 9 h. The effects of both epinephrine and cortisol on protein synthesis are also much larger in cells from adrenalectomized animals. After a delay of several hours, cortisol increases fibrinogen synthesis sharply, so that at the end of the 20-h incubation, cells treated with hormone have secreted nearly 2.5 times as much fibrinogen as control cells. The effect is specific; cortisol stimulates neither albumin secretion nor intracellular protein synthesis. The combination of cortisol and epinephrine strongly depresses albumin synthesis in both types of cells. Insulin enhances albumin and general protein synthesis but has little effect on fibrinogen synthesis.

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