Effect of gamma globulin on reactivity of the organism. V. Types of biological activity of gamma globulin preparations]

Biological activity of 110 series of commercial gamma-globulin preparations was studied; they were found to contain placental antigens, group-specific blood substances, gonadotropic hormones and antibodies to them. Placental antigens were found in 12% of placental and abortive gamma-globulin batches in titres of 1 : 2--1 : 16; no placental protein was revealed in donor gamma-globulin. There were group-specific blood substances in all the batches of placental and abortive gamma-globulin studied (in titres of 1 : 138--A, 1 : 112 B in the placental gamma-globulin and in titres of 1 : 48.9--A, 1 : 32--B in the abortive gamma-globulin). In the preparations from the venous blood group-specific substances were either absent or present in lowe titres only (1 : 2). The value of gonadotropic hormones in the placental gamma-globulin batches constituted 873+/-157, and in the abortive--991.4+/-147 IU/l; no gonadotropins were revealed in donor gamma-globulin. The mean titres of antibodies to gonadotropin hormone in the gamma-globulin preparations made of placental blood constituted 1 : 236+/-32, of abortive--1 : 131+/-16.6, and of the venous blood--1 : 46+/-24.7. The presence of biologically-active substances in the gamma-globulin preparations pointed to the necessity of increased requirement of their quality; additional requirements to its standardization proved to be also necessary.     

Studies on the proteolytic activity of γ-globulin preparations

1. The proteolytic activities of several gamma-globulin preparations were tested. These included sulphate-precipitated human and bovine preparations and human and bovine Cohn fraction II preparations as well as purified gamma-globulin preparations. Up to 14mg. of diffusible peptides and glycopeptides/g. of gamma-globulin was liberated after dialysis and up to 10mg. of peptides/g. after incubation and trichloroacetic acid precipitation, as products of the degradation process in incubated gamma-globulin. 2. in-Aminohexanoic acid and p-chloromercuribenzoic acid, as well as heating at 60 degrees for 40min., were shown to inhibit strongly these proteolytic activities. Streptokinase was shown to activate strongly the proteolytic activity of all the human preparations (sulphate-precipitated, Cohn fraction II, and purified gamma-globulin). 3. Two distinct pH optima were shown for human and bovine gamma-globulin preparations: one at pH8, the other at pH3.8 (the latter activity could be demonstrated only in the presence of cysteine). 4. Both (131)I-labelled human Cohn fraction II and bovine fibrinogen were attacked by a sulphate-precipitated preparation of gamma-globulin. Of the synthetic substrates tested toluene-p-sulphonyl-l-arginine methyl ester was hydrolysed by both the sulphate-precipitated and Cohn fraction II preparations, as was benzoyl-l-arginine amide at pH5, but only in the presence of cysteine. 5. These data are interpreted to indicate that at least two enzymes are present in gamma-globulin preparations, one being similar to the plasmin system, the other similar to cathepsin B.     

Proteolysis of products of mitochondrial protein synthesis in isolated mitochondria of Saccharomyces cerevisiae yeasts]

Products of mitochondrial protein synthesis were specifically labeled with 3H-leucine in the presence of cycloheximide at the end of the exponential phase of yeast aerobic growth on glucose. The mitochondria isolated from these cells lost 37-40% of the label from the protein fraction during 60 min incubation at 35 degrees, which was accompanied by the accumulation of 3H-leucine in TCA-soluble fraction. This process was suppressed by phenyl-methyl sulfonyl fluoride and p-chloromercuriphenyl sulfonate, the inhibitors of proteases, and could thus be considered as the proteolysis of the products of mitochondrial protein synthesis. The proteolysis was ATP dependent and was stimulated by puromycine which is known to induce the removal of incomplete polypeptides from mitochondrial ribosomes. A body of indirect evidence allows a suggestion to be made that the observed proteolysis can hardly be due to the action of cytoplasmic proteinases.     

Biosimilarity under stress: A forced degradation study of Remicade® and Remsima™

Remsima? (infliximab) is the first biosimilar monoclonal antibody (mAb) approved by the European Medical Agency and the US Food and Drug Administration. Remsima? is highly similar to its reference product, Remicade®, with identical formulation components. The 2 products, however, are not identical; Remsima? has higher levels of soluble aggregates, C-terminal lysine truncation, and fucosylated glycans. To understand if these attribute differences could be amplified during forced degradation, solutions and lyophilized powders of the 2 products were subjected to stress at elevated temperature (40–60°C) and humidity (dry-97% relative humidity). Stress-induced aggregation and degradation profiles were similar for the 2 products and resulted in loss of infliximab binding to tumor necrosis factor and FcγRIIIa. Appearances of protein aggregates and hydrolysis products were time- and humidity-dependent, with similar degradation rates observed for the reference and biosimilar products. Protein powder incubations at 40°C/97% relative humidity resulted in partial mAb unfolding and increased asparagine deamidation. Minor differences in heat capacity, fluorescence, levels of subvisible particulates, deamidation and protein fragments were observed in the 2 stressed products, but these differences were not statistically significant. The protein solution instability at 60°C, although quite significant, was also similar for both products. Despite the small initial analytical differences, Remicade® and Remsima? displayed similar degradation mechanisms and kinetics. Thus, our results show that the 2 products are highly similar and infliximab's primary sequence largely defines their protein instabilities compared with the limited influence of small initial purity and glycosylation differences in the 2 products.     

The effect of hydrocarbon addition on the deamidation rate in immune whey proteins

The influence of some hydrocarbons that are often used at different stages of immunobiological preparation's production as stabilizers of biological activity on the dynamics of nonenzymatic deamidation in proteins of immune whey against conditionally pathogenic microorganisms obtained by means of membrane ultrafiltration technology is investigated. Preparations of whey were incubated in 10 per cent solutions of glucose, fructose and sorbitol at the conditions similar to physiological ones (0.9% NaCl, pH 5.5) and temperature of about +4 degrees C and +35 degrees C for 7, 14 and 28 days. A sample dissolved in 0.9% NaCl (pH 5.5) without addition of hydrocarbons was used as a "control preparate". All explored substances brought about the suppressive effect on deamidation rate of asparaginyl residues whereas that of glutaminyl residues, on the contrary, was obviously increased. The possible reasons for these observations are discussed.     

Safety of human blood products: inactivation of retroviruses by heat treatment at 60 degrees C

Acquired immune deficiency syndrome (AIDS) can be transferred to patients by blood transfusions or human blood preparations, such as cryoprecipitates or factor VIII concentrates. Retroviruses have been discussed as infectious AIDS agents and more recently human T-lymphotropic retroviruses designated as HTLV type III and LAV (lymphadenopathy-associated virus) have been isolated from AIDS patients. Whether heat treatment at 60 degrees C (pasteurization) of liquid human plasma protein preparations inactivates retroviruses was therefore investigated. Pasteurization had already been included in the routine manufacturing process of human plasma protein preparations in order to guarantee safety with regard to hepatitis B. Since high titer preparations of human retroviruses were not available, heat inactivation was studied using Rous sarcoma virus added to the various plasma protein preparations tested. This retrovirus which was obtained in preparations of 6.0 log10 FFU/ml was shown to be at least as heat stable as two mammalian retroviruses studied, i.e., feline and simian sarcoma virus. In all of eight different plasma protein preparations tested, Rous sarcoma virus was completely inactivated after a heat treatment lasting no longer than 4 hr. It is thus concluded that pasteurization of liquid plasma protein preparations at 60 degrees C over a period of 10 hr must confer safety to these products with respect to AIDS, provided that the AIDS agents are retroviruses of comparable heat stability as Rous sarcoma virus and the mammalian retroviruses tested.     

The concentration of antibodies to several exoenzymes of beta-hemolytic streptococci in commercial preparations of gamma globulin]

For the purpose of possible use of commercial preparations of homologous gamma-globulin in the outbreaks of streptococcus affections the authors studied the content of antibodies to some streptococcus antigens, namely to O-streptolysin, streptohyaluronidase and streptokinase. High antibody titres to streptokinase were observed in 62.7, to O-streptolysin--in 35.2 and to streptohyaluronidase--in 15.4% of the batches under study. The greatest amount of antibodies to all the antigens under study was revealed in gamma-globulin prepared from the blood sera collected during the automno-winter period of the year.     

Proteolytic activity of arginine esterase from dog seminal plasma towards actin and other structural proteins. Comparison with trypsin and kallikrein

At equimolar ratio of enzyme/substrate, actin, tropomyosin, fibronectin and myosin were extensively hydrolyzed during an incubation of one hour at 37 degrees C. Dog serum albumin, ovalbumin, bovine gamma-globulin and human prostatic acid phosphatase were not hydrolyzed. The activity of arginine esterase towards actin at pHs 6.5, 7.1 and 7.6 was respectively 60, 74 and 84% of the one found at optimum pH 8.2. The cleavage products of actin by arginine esterase and trypsin were similar although trypsin activity was 5000-fold higher. Kallikrein produced a major fragment of actin not observed with arginine esterase and trypsin. It is concluded that arginine esterase has a low trypsin-like activity towards structural proteins and that this activity may have a physiological significance.     

Physiological responses of hybridoma cells in a protein-free medium to soluble and immobilized antigen

This study examined the effect of antigen in a protein free medium on cell growth and monoclonal antibody production by a hybridoma line. Antigen immobilized on a Sepharose gel matrix via a bovine gamma-globulin carrier protein was used to stimulate the cell cultures in T-flasks. In comparison to antigen-free culture, total antibody production during was increased up to 40%, while slower cell growth rates were observed. The specific antibody production during the stationary culture phase was 40% to 80% higher in the presence of immobilized antigen. The surface density of antigen on the Sepharose beads had a strong influence on the physiological response of the hybridomas. (c) 1994 John Wiley & Sons, Inc.     

Nutrient medium for the isolation of hemocultures from brucellosis patients]

To isolate brucellosis causative agent from the blood of patients against the background of antibiotic therapy the authors used for the first time the medium consisting of aminopeptide, beta-globulin agar with glucose, glycerine, and twin-60. No valuable food products--meat, liver--were required to prepare the medium; by-product of gamma-globulin production being used. With the aid of the suggested medium there was isolated 2.4 times more hemocultures than on the widely used meat-peptone medium. This is attributed to the presence in the medium of human protein, essential amino acids, and blood mineral substances, which, in combination, facilitated adaptation of brucellosis causative agent to the nutrient medium similar to human blood by composition. Economical effect in using the mentioned medium constituted 53.8% annually.     

Evidence that the Rous sarcoma virus transforming gene product is associated with glycerol kinase activity

This communication provides biochemical, immunological, and genetic evidence that pp60src, the Rous sarcoma virus transforming gene product, is associated with glycerol kinase activity. Our investigations demonstrated that the compound phosphorylated by pp60src or by glycerol kinase (EC 2.7.1.30) from Candida mycoderma share the same electrophoretic and chromatographic mobilities. The glycerol kinase and protein kinase activities of pp60src were inhibited similarly by preincubation with immune IgG. Both activities were reduced 6-9-fold in pp60src preparations derived by immunoaffinity chromatography from cells which were infected with NY68, a temperature-sensitive transformation mutant of Rous sarcoma virus. The thermolability at 41 degrees C of the glycerol kinase activity of pp60src from the mutant virus-infected cells was greater (t/2 = 1.3 min) than the same activity in pp60src preparations from wild type virus-infected cells (t/2 = 4.8 min).     

Thermobarostability of alpha-chymotrypsin in reversed micelles of aerosol OT in octane solvated by water-glycerol mixtures

Thermostability of alpha-chymotrypsin at normal pressure in reversed micelles depends on both an effective surfactant solvation degree and glycerol content in the system. The difference in alpha-chymotrypsin stability in reversed micelles at various glycerol concentrations [up to 60% (v/v)] was more pronounced at high surfactant degrees of solvation, R >/= 16. After a 1-h incubation at 40 degrees C in "aqueous" reversed micelles (in the absence of glycerol), alpha-chymotrypsin retained only 1% of initial catalytic activity and 10, 22, 59, and 48% residual activity in glycerol-solvated micelles with 20, 30, 50, and 60% (v/v) glycerol, respectively. The explanation of the observed effects is given in the frames of micellar matrix structural order increasing in the presence of glycerol as a water-miscible cosolvent that leads to the decreasing mobility of the alpha-chymotrypsin molecule and, thus the increase of its stability. It was found that glycerol or hydrostatic pressure could be used to stabilize alpha-chymotrypsin in reversed micelles; a lower pressure is necessary to reach a given level of enzyme stability in the presence of glycerol.     

Changes in Antioxidative Properties of Lactoferrin from Women's Milk during Deamidation

Spontaneous deamidation of lactoferrin preparations from women's milk was found during incubation for 28 days under simulated physiological conditions (0.85% NaCl, pH 7.0, 37°C). After 28 days of incubation, this deamidation was associated with a 12% decrease in the total amide content in the protein. Addition of deamidated preparation to a suspension of lipoproteins from egg yolk in the presence of Rhodamine 6G decreased the total intensity of rapid and slow emission and also the intensity of the slow emission of Fe²⁺-induced chemiluminescence by 37, 48, and 53%, respectively, suggesting an increase in the antioxidative activity of lactoferrin during deamidation. Deamidation obviously stimulated the nonspecific interaction of lactoferrin with iron ions and, consequently, increased the antioxidant effect of the protein in this way. This was supported by the finding of decreased antioxidative effectiveness of lactoferrin during its complete saturation with iron under the incubation conditions.     

Low-temperature microbial degradation of crude oil products differing in the extent of condensation

Out of the 30 strains capable of oil degradation at 4-6 degrees C, four were selected by the ability to degrade 40% of the oil substrate present in the growth medium: Rhodococcus spp. DS-07 and DS-21 and Pseudomonas spp. DS-09 and DS-22. We studied the activity of these strains as degraders of oil products of various condensation degrees (crude oil, masut, petroleum oils, benzene resins and ethanol-benzene resins) at 4-6 degrees C. The maximum degrees of degradation of masut and ethanol-benzene resins were observed in Pseudomonas spp. DS-22 (17.2% and 5.2%, respectively). The maximum degradation of petroleum oils and benzene resins was observed in Rhodococcus spp. DS-07 (40% and 16.6%, respectively). The strains provide a basis for developing biodegrader preparations applicable to bioremediation of oil-polluted sites under the conditions of cold climate.     

Effect of protein conformation on rate of deamidation: ribonuclease A

The effect of the folded conformation of a protein on the rate of deamidation of a specific asparaginyl residue has been determined. Native and unfolded ribonuclease A (RNase A) could be compared under identical conditions, because stable unfolded protein was generated by breaking irreversibly the protein disulfide bonds. Deamidation of the labile Asn-67 residue of RNase A was followed electrophoretically and chromatographically. At 80 degrees C, similar rates of deamidation were observed for the disulfide-bonded form, which is thermally unfolded, and the reduced form. At 37 degrees C and pH 8, however, the rate of deamidation of native RNase A was negligible, and was more than 30-fold slower than that of reduced, unfolded RNase A. This demonstrates that the Asn-67 residue is located in a local conformation in the native protein that greatly inhibits deamidation. This conformation is the beta-turn of residues 66-68.     

Effect of polyols on the conformational stability and biological activity of a model protein lysozyme

The purpose of this study was to investigate the stabilizing action of polyols against various protein degradation mechanisms (eg, aggregation, deamidation, oxidation), using a model protein lysozyme. Differential scanning calorimeter (DSC) was used to measure the thermodynamic parameters, mid point transition temperature and calorimetric enthalpy, in order to evaluate conformational stability. Enzyme activity assay was used to corroborate the DSC results. Mannitol, sucrose, lactose, glycerol, and propylene glycol were used as polyols to stabilize lysozyme against aggregation, deamidation, and oxidation. Mannitol was found to stabilize lysozyme against aggregation, sucrose against deamidation both at neutral pH and at acidic pH, and lactose against oxidation. Stabilizers that provided greater conformational stability of lysozyme against various degradation mechanisms also protected specific enzyme activity to a greater extent. It was concluded that DSC and bioassay could be valuable tools for screening stabilizers in protein formulations.     

Effects of solution polarity and viscosity on peptide deamidation.

Deamidation kinetics were measured for a model hexapeptide (L-Val-L-Tyr-L-Pro-L-Asn-Gly-L-Ala, 0.02 mg/mL) in aqueous solutions containing glycerol (0-50% w/w) and poly(vinyl pyrrolidone) (PVP, 0-20% w/w) at 37 degrees C and pH 10 to determine the effects of solution polarity and viscosity on reactivity. The observed pseudo-first order deamidation rate constants, k(obs), decreased markedly when the viscosity increased from 0.7 to 13 cp, but showed no significant change at viscosities >13 cp. Values of k(obs) also increased with increasing dielectric constant and decreasing refractive index. Molecular dynamics simulations indicated that the free energy associated with Asn side-chain motion is insensitive to changes in dielectric constant, suggesting that the observed dielectric constant dependence is instead related primarily to the height of the transition state energy barrier. An empirical model was proposed to describe the effects of the viscosity, refractive index and dielectric constant on k(obs). Analysis of the regression coefficients suggested that both permanent and induced dipoles of the medium affect the deamidation rate constant, but that solution viscosity is relatively unimportant in the range studied.     

Increased thermostability of Asn182 --> Ala mutant Aspergillus awamori glucoamylase

Asn182 --> Ala Aspergillus awamori glucoamylase expressed in Saccharomyces cerevisiae had a first-order thermodeactivation coefficient 40% that of wild-type glucoamylase at pH 4.5 between 60 degrees and 65 degrees C, caused by the elimination of an Asn-Gly sequence subject to deamidation and eventual chain breakage. Above 70 degrees C, and at pHs 3.5 and 5.5, thermodeactivation coefficients of wild-type and mutant enzymes were roughly equal, because the fastest deactivation mechanism was no longer deamidation. The mutation had little effect on the enzyme's optimal pH for activity and subsite map, or on the glucose yield from starch dextrin hydrolysis. During enzyme production by yeast fermentation, highest cell densities and activities of wild-type and mutant glucoamylases were attained after a period of glucose starvation, followed by a second addition of glucose. (c) 1994 John Wiley & Sons, Inc.     

Effects of amino acid sequence, buffers, and ionic strength on the rate and mechanism of deamidation of asparagine residues in small peptides

The nonenzymatic rates of deamidation of Asn residues in a series of pentapeptides with the sequences VSNXV and VXNSV, where X is one of 10 different amino acids, were determined at neutral, alkaline, and acid pH values. The results demonstrate that in neutral and alkaline solutions the amino acid residue on the amino side of the Asn had little or no effect on the rate of deamidation regardless of its charge or size. The group on the carboxyl side of Asn affected the rate of deamidation significantly. Increasing size and branching in the side chain of this residue decreased the rate of deamidation by as much as 70-fold compared to glycine in the N-G sequence, which had the greatest rate of deamidation. In acidic solution, the rate of deamidation of the Asn residue was not affected by the amino acid sequence of the peptide. The products for each deamidation reaction were tested for the formation of isoAsp residues. In neutral and alkaline solutions, all products showed that the isoAsp:Asp peptide products were formed in about a 3:1 ratio. In acidic solution, the Asp peptide was the only deamidation product formed. All peptides in which a Ser residue follows the Asn residue were found to undergo a peptide cleavage reaction in neutral and alkaline solutions, yielding a tripeptide and a dipeptide. The rate of the cleavage reaction was about 10% of the rate of the deamidation pathway at neutral and alkaline pH values. The rates of deamidation of Asn residues in the peptides studied were not affected by ionic strength, and were not specific base catalyzed. General base catalysis was observed for small bases like ammonia. A model for the deamidation reaction is proposed to account for the observed effects.     

Deamidated active intermediates in the irreversible acid denaturation of ribonuclease-A.

A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH less than 1) acqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromotographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa1. The conversion of RNase-A to RNase-Aa1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa1 was found to have very nearly the same activity and physicochemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.