J. James Sedmak

A rapid, sensitive, and versatile assay for protein using Coomassie brilliant blue G250

Abstract An assay for proteins in solution is described that depends on the conversion of Coomassie brilliant blue G250 in dilute acid from a brownish-orange to an intense blue color. As soon as the dye solution is mixed with a protein sample, the absorbance of the mixture can be measured. The absorbance of the solution is stable for 60–90 min at room temperature. The test can be used with a variety of proteins and polypeptides with molecular weights greater than 3000. The assay has high reproducibility and can detect less than 1.0 μg of albumin. The single reagent required is very stable, and free amino acids and several chemicals that interfere with the Lowry protein assay cause no interference. The test is adaptable to a micromethod, which is also described.

Transport of γ-interferon into the cell nucleus may be mediated by nuclear membrane receptors

Abstract Purified mouse interferon gamma (MuIFN-γ), a lymphokine having potent antiviral, immunomodulatory, and growth inhibitory activities is internalized ( t 1 2 ) by mouse L929 fibroblasts via receptor-mediated endocytosis. Individual MuIFN-γ molecules, identified by a postembedding immuno-gold technique, are then transported to the cell nucleus, perhaps through nuclear pores, into areas of dense chromatin. Purified, isolated nuclei of L929 cells bind radiolabeled MuIFN-γ specifically and with high affinity (Kd = 2×10−10 M). These nuclear membrane receptors, distinct from those for MuIFN-β, number about 24,000/nucleus. Treatment of nuclei with trypsin prevents binding of MuIFN-γ. The demonstration of rapid cellular uptake and transport of MuIFN-γ into the dense chromatin, perhaps facilitated by nuclear receptors, suggests that IFN-γ molecules, alone or bound to receptor, may directly affect genome regulation.

Lanthanide ion enhancement of interferon binding to cells

Pretreatment of purified [125I]-labeled human and mouse beta interferons (IFN) with lanthanum chloride (LaCl3) enhanced 20-30 fold the binding of the [125I]-IFNs to human A549 and mouse L cells at 0 degree C and also enhanced antiviral activity in homologous cells. Although lanthanides enhanced cross-species binding of both human and mouse [125I]-IFNs, there was no increase in cross-species antiviral activity. Unlabeled IFN not treated with LaCl3 did not compete with [125I]-IFN treated with LaCl3 for cellular receptors. However, unlabeled IFN treated with LaCl3 did compete with LaCl3-treated [125I]-IFN. These results suggest that lanthanide treated IFNs do not bind to the same receptors as native IFNs.

Interferon Stabilization and Enhancement by Rare Earth Salts

The addition of salts of the rare earth elements (lanthanides) enhances by a factor of two or more the initial activity of human fibroblast, leukocyte and immune interferons as well as mouse L-cell interferon. Furthermore, the salts of the lanthanides (0.01 to 0.002 M) preserve the activity of human fibroblast interferon not only after 4 days of heating at 37 degrees C but also after exposure to severe shearing forces; human leukocyte interferon was stabilized to a lesser degree, albeit significantly.

Interferon bioassay: reduction in yield of myxovirus neuraminidases.

Summary Interferon reduced the production of virus neuraminidase during single-cycle growth of A2/HK/1/68 or recombinant X7(F1) influenza viruses in chicken embryo cell cultures. Neuraminidase activity, measurable within 6 h after infection, was reduced as much as 70 to 80% below control levels in interferon-treated cultures. X7(F1) neuraminidase yield was at least as sensitive a measure of interferon inhibition as vesicular stomatitis virus in the standard plaque-reduction assay. Interferon titre, expressed as the 30% neuraminidase reduction dose or NRD30, is derived from semi-logarithmic sigmoidal dose—response curves. The advantages that recommend the neuraminidase reduction bioassay for interferon assay include the precision of the enzyme assay (±1.0%), the precision of measurement of replicate samples of interferon (±9%), the reproducibility of interferon titres obtained sequentially (±20%), as well as the rapidity and economy of the method.

The Neuraminidase Yield-Reduction Bioassay of Human and Other Interferons

Summary The production of neuraminidase by the recombinant influenza virus X7(F1) in human, monkey, rabbit, hamster, mouse, and chicken cell cultures is inhibited by interferon. Described is a new enzyme assay for neuraminidase that can be applied to the bioassay of interferons. The advantages of this interferon bioassay are its sensitivity, reproducibility, rapidity, and convenience. We thank Mary Dixon, Joyce F. Otto, and Christine Schoenherr for their excellent technical assistance. Note added in proof: Experiments subsequent to this manuscript have revealed that the production of neuraminidase in the RK-13 cell line is variable and at times too low for use in an interferon assay. Note added in proof: Low yields of neuraminidase have recently been obtained in the RK-13 rabbit cell line, an observation which makes this line unsuitable for the neuraminidase bioassay. However, high neuraminidase yields are obtained in another interferon-sensitive line of rabbit skin (RS) cells.

The cellular internalization of recombinant gamma interferon differs from that of natural interferon gamma.

Purified natural and recombinant murine gamma interferons (MuIFN-gamma) bind at 4 degrees C to cultured L929 mouse fibroblasts with comparable receptor-binding affinity (Kd = 9 x 10(-10) M). Both 125I-labeled MuIFNs are rapidly internalized by cells at 37 degrees C, although recombinant IFN is internalized somewhat more slowly than natural IFN (t1/2 = 90 sec and 45 sec, respectively). Immunoelectronmicroscopy showed that the majority of bound recombinant MuIFN-gamma was located on the plasma membrane outside of coated areas, whereas natural interferon was found mainly in coated pits. At 37 degrees C most of the recombinant molecules entered the cytoplasm in pinocytotic vesicles, while natural interferon was internalized by the specific mechanism of receptor-mediated endocytosis [1]. However, nearly equal amounts of immunocytochemically detectable molecules of both IFNs were found in the cell nucleus within 2-3 min incubation at 37 degrees C. Thus, the process of translocation of the recombinant IFN-gamma appears to differ from that of the natural product.

[83] Procedures for stabilization of interferons

Publisher Summary This chapter discusses the procedures for stabilization of interferons. The stability of interferons, especially as they are processed and purified, has proved to be a greater problem than is appreciated from early observations on the stability of crude materials. It is now realized that interferons in solution can be inactivated by a variety of physical and chemical treatments, but a degree of stability can be provided by means of certain additives. For long-term storage of interferon preparations, particularly those intended for clinical use, freeze-drying is preferred. One of the most effective methods of preserving interferon activity for extended periods, such as storage of reference reagents or preparations for clinical use, is freeze-drying in the presence of low concentrations of added protein and a sodium phosphate buffer. Two accelerated storage tests are used to predict the stability of freeze-dried interferons. The accelerated multiple isothermal stability test is used to provide a conservative estimate of the expected stability of these standards under long-term storage conditions.

[53] Virus yield-reduction assays for interferon with the influenza virus neuraminidase assay

Publisher Summary This chapter describes virus yield-reduction assays for interferon with the influenza virus neurarninidase assay. The measure of reduction in yield of influenza A virus neuraminidase in infected cell cultures is a sensitive and reproducible bioassay for various interferons. A recombinant virus X7(F 1 ) (HON2) 3 is the most interferon sensitive of 12 influenza viruses tested and also produced higher yields of neuraminidase in most cell cultures than the other influenza viruses. This recombinant virus produces neuraminidase even in some cells that fail to produce infectious virus, but the best yields of neuraminidase were in primary chicken embryo cell cultures, which do produce infectious virus. The production of X7(F 1 ) neuraminidase is measured by the thiobarbituric acid method with the glycoprotein fetuin as substrate or by a method with the synthetic substrate methoxyphenylneuraminic acid.

Thermal and vortical stability of purified human fibroblast interferon.

The loss of biological activity upon heating or agitation of human interferons is markedly altered by changing their aqueous environment. Low pH significantly stabilizes liquid fibroblast interferon at 68 degrees C and 37 degrees C whereas chaotropic salts stabilize at 68 degrees C but not at 37 degrees C; this anomalous result may be due to reactivation of biological activity at the higher temperature. The concentration of extraneous proteins influences the apparent thermal stability at any temperature and pH; thus, interferon was not stable even at low pH at protein concentrations less than 5 microgram/ml. Solutions of partially purified fibroblast interferon can be inactivated by mechanical stress; the addition of proteins or nonionic detergents prevents such inactivation. Freeze-dried preparations show the greatest thermal stability. The use of high-temperature, accelerated storage tests makes it possible to predict the shelf-life of freeze-dried interferon.