Comparative effects of stabilizing additives on the rates of heat inactivation of recombinant human interferon alpha-2b in solution.

This study was aimed at the comparative evaluation of stabilizing additives used for the protection of the antiviral activity of interferon-alpha2b against thermal inactivation, at 60 degrees C. The comparative effects of amino acids, polyhydric alcohols, saccharides and nonionic surfactants were studied. All were effective. Representing the thermal inactivation process with first order kinetics, a maximal prolongation of antiviral activity half-life of 39-fold was achieved with the most effective procedure. Inactivation rate constants varied from (53.3+/-4.6)x10(-3) to (2.5+/-0.3)x10(-3) min(-1). Human serum albumin, nonionic surfactants and monosaccharides increased half-life values by 5-39-, 5-23-, 4-20-fold, respectively. Amino acids, polyhydric alcohols and disaccharides increased t(1/2) values by 4-11-, 2-8- and 3-8-fold, respectively. These data provide useful information for the selection of stabilizing additives for IFN-alpha2b formulations.

Determination of first and second derivatives of progress curves in the case of unknown experimental error.

The first and second derivatives of progress curves are obtained from the cubic spline function. The new approach is based on a development of the splining quality test which was used for estimating the precision of the splining. The proposed method is used on a computer with a FORTRAN 77 program. The method may also be applied for an approximate estimation of experimental error.

[Determination of the enzymatic activity of lysine amidase]. / Opredelenie fermentativnoi aktivnosti lizinamidazy.

A technique is proposed for determining lysinamidase and aminolactamase activities of lysinamidase (EC 3.5.1.n.). It is based on spectrophotometric measurement of the optical density decrease of the substrate solution at 227 nm. For cyclic lysinamide L-alpha-amino-epsilon-caprolactam epsilon 227 M = 151 M-1.cm-1, for linear lysinamide epsilon 227 M = 73 M-1.cm-1, and for lysine epsilon 227 M = 5 M-1.cm-1. The technique is simple and requires no additional reagents.

Determination of initial velocities of enzymic reactions from progress curves.

The present communication describes a novel method for estimating initial velocities (v) of enzyme-catalysed reactions. It is based on an approximation of experimental data obtained by the cubic spline function. The initial velocity of a reaction is calculated as a derivative of the approximating function at a time value equal to zero. The proposed method is usable on a computer with a FORTRAN IV program. The method can be successfully used in such cases as substantial extents of substrate conversion, the inactivation of an enzyme in the course of a reaction, the existence of large experimental error or when the reaction mechanism is unknown.

Lysine decarboxylase assay by the pH-stat method.

A simple assay of lysine decarboxylase is described. It is based on measuring the rate of titration of OH- ions, released during decarboxylation with the aid of a pH-stat apparatus. The continuous recording of the reaction progress may be easily performed. Using the pH-stat method the Km for lysine decarboxylase from E. coli is 2.0 mM.

[Choice of the initial enzyme correlation in immobilizing polyenzyme systems]. / Vybor iskhodnogo sootnosheniia fermentov pri immobilizatsii polifermentnykh sistem.

An optimal, in respect to productivity (activity X stability), enzyme ratio for immobilization of multienzyme systems was calculated by using the kinetic parameters (KM and Vmax), data on the stability and yield of each enzyme during immobilization. The experimental data, obtained during combined immobilization of invertase, mutarotase and glucose oxidase, illustrate the theoretical propositions.

[Determination of beta-mannanase activity by viscosimetric and spectrophotometric methods]. / Opredelenie aktivnosti beta-mannanazy viskozimetricheskim i spektrofotometricheskim metodami.

The activity of beta-mannanase from Bacillus subtilis was measured viscosimetrically and spectrophotometrically. As substrate galactomannane of Ceratonia siliqua was used. Relationships between the beta-mannanase activity and the substrate concentration as well as the enzyme content were investigated. The kinetic parameters of the enzymes obeying the Michaelis-Menten equation were calculated. It was found viscosimetrically that Vmax of the commercial enzyme preparation was 1.4 mucat/g (at pH 5.8 and 40 degrees) and Km was 0.6 mM. The viscosimetric method shows high sensitivity, whereas the spectrophotometric technique suits mass-scale analyses.

[Isolation and characteristics of immobilized L-glutamate decarboxylase]. / Poluchenie i kharakteristika immobilizovannoi L-glutamatdekarboksilazy.

Glutamate decarboxylase from Escherichia coli was immobilized on inorganic macroporous carriers by the glutaraldehyde, carbodiimide and bromacetyl methods, on silicagel coated with a layer of a glutaraldehyde and m-phenylene diamine copolymer, and by polyacrylamide gel incorporation. The efficiency of the above methods of immobilization was evaluated. The bromacetyl method was found to be the most efficient. The dependence of activity of soluble and immobilized Glu-decarboxylase upon pH, temperature, substrate concentration, and stability was established. The differences in the properties of soluble and immobilized Glu-decarboxylase were due to the substrate diffusion in pores of the carrier. The immobilized Glu-decarboxylase obtained showed high activity and stability.

[Preparation and charaterization of aminopolysterol immobilized invertase]. / Poluchenie i svoistva invertazy, immobilizovannoi na aminopolistirole.

Invertase from Saccharomyces cerevisiae was immobilized on aminopolysterol by adsorption, glutaraldehyde, carbodiimide or bromacetyl methods. The dependence of activity of invertase immobilized by the carbodiimide method upon pH, temperature and sucrose concentration was studied. The "effective" Michaelis constant of the immobilized preparation was 6 times higher than that of soluble inverase. At high sucrose concentrations (beginning with 0.2 M for immobilized and 0.9 M for soluble invertase) substrate inhibition was observed. The data on stability of immobilized invertase during refrigerated storage and in a working flow column were obtained.

[Thermostability of glucose oxidase immobilized on silica gel by different methods]. / Termostabil'nost' gliukozooksidazy, immobilizovannoi na silikagele razlichnymi, metodami.

Immobilization and thermostability of glucose oxidase immobilized on silica gel MCA-750 7 by different methods were studied. The process of inactivation was found to follow two stages that differed in their rate. The most stable preparations were produced by immobilization based on bromacetyl and glutaraldehyde methods.