scholarly journals Improvement of the method of obtaining human IgA Fc-fragments

2015 ◽  
Vol 6 (1) ◽  
pp. 32-35
Author(s):  
O. Y. Galkin ◽  
Y. V. Gorshunov ◽  
V. F. Solovjova
Keyword(s):  
Enzymatic Hydrolysis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Nitrogen Atmosphere ◽  
Maximum Output ◽  
Serological Tests ◽  
Isolation And Purification ◽  
Antibody Conjugate ◽  
Hydrolysis Of

To address a number of fundamental and applied problems in immunology, molecular and cellular biology and biotechnology it is necessary to obtain Fc-fragments of immunoglobulins. Fc-fragments may be used for studying of the effector functions of antibodies which are mediated by these areas. They are often used as an immunogen to produce anti-specie (based on so-called secondary antibody) conjugate in the development of serological tests for diagnostics (predominantly such conjugate based on monoclonal antibodies). The work is aimed to develop improved methods of obtaining and allocation of Fc-fragments of human IgA. To achieve this objective, optimization of hydrolysis of IgA with subsequent purification of Fс-fragments have been carried out. Improved method of obtaining Fc-fragments of IgA provides: papain hydrolysis of immunoglobulin in the environment of nitrogen for 4 h, allowing to achieve maximum output of Fc-fragments without their further degradation: isolation and purification of Fc-fragments of human IgA by one-stage gel filtration on sephadex G-100; control of purity of the target product by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate and Ouchterlony immunodiffusion. Enzymatic hydrolysis was carried out at the optimal temperature of papain (37 °C). As the oxygen in the air may have inhibitory effect on enzymatic hydrolysis reaction, the reaction mixture was incubated in the nitrogen atmosphere to prevent inactivation of papain. To reduce the incident degradation of immunoglobulin molecules, papain hydrolysis was carried out without using an enzyme activator (cysteine). Usage of the proposed scheme allows obtaining Fc-fragments of human IgA of high purity. Outcome of Fc-fragments after all stages of purification was about 18% of the initial amount of IgA in the preparation. Molecular weight from Fc-fragments of human IgA was equal to approximately 70 kDa.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

scholarly journals Anti-coagulant Activity of Isolated and Partially Characterized Proteoglycans and Glycosaminoglycans from African Night Crawler (Eudrilus eugeniae Kinberg)

KIMIKA ◽  
2015 ◽  
Vol 26 (2) ◽  
pp. 31-38
Author(s):  
Mia Clare Marie L. Bercansil ◽  
Miko Lorenzo J. Belgado
Keyword(s):  
Hyaluronic Acid ◽  
Infrared Spectroscopy ◽  
Enzymatic Hydrolysis ◽  
Gel Filtration ◽  
Acetate Buffer ◽  
Eudrilus Eugeniae ◽  
Gel Filtration Chromatography ◽  
Molecular Weights ◽  
Coagulant Activity ◽  
Hydrolysis Of

Proteoglycans and glycosaminoglycans were isolated from African night crawler (Eudrilus eugeniae Kinberg) and partially characterized proteoglycans (3.04 % of lyophilized worm) were liberated from the defatted and depurinated worm samples by dissociative method using 4M urea in acetate buffer. Glycosaminoglycans (12.47% of proteoglycan extract) were extracted using enzymatic hydrolysis of the proteoglycan extract with papain. Gel filtration chromatography using Sepharose CL-4B was used to purify and estimate the molecular weights of the proteoglycan and glycosaminoglycan fractions. Three proteoglycan fractions PGF1, PGF2 and PGF3 with estimated molecular weigths 860 kDa, 181 kDa and 3 kDa, respectively were identified as monitored by the Bradford and modified carbazole assay. Two glycosaminoglycan fractions - GF1 (MW = 860 kDa) and GF2 (MW=140 kDa) were identified using the modified carbazole assay. Infrared spectroscopy of the GF1 and GF2 showed the possible identities of the fractions. GF1 may be a hyaluronic acid and GF2 is possibly chondroitin. Anti-coagulant assay for the extracts and fractions revealed that the glycosaminoglycan isolate has anti-coagulant activity but not the GF1 and GF2 fractions individually.

scholarly journals The enzymatic method for the quantitative determination of benzalkonium chloride in the antiseptic solution “CUTASEPT® F”

2021 ◽  
Vol 19 (4(76)) ◽  
pp. 33-39
Author(s):  
Olena V. Koval’ska ◽  
Mykola Ye. Blazheyevskіy
Keyword(s):  
Enzymatic Hydrolysis ◽  
Quantitative Determination ◽  
Peracetic Acid ◽  
Benzalkonium Chloride ◽  
Chloride Content ◽  
Inhibitory Effect ◽  
Enzymatic Method ◽  
Limit Of Quantitation ◽  
Hydrolysis Of

Aim. To develop an alternative method for the quantitative determination of the benzalkonium chloride content as an active pharmaceutical ingredient in the disinfectant solution “CUTASEPT® F”.Materials and methods. The method is based on the ability of benzalkonium chloride to inhibit the enzymatic hydrolysis of acetylcholine by acetylcholinesterase. The reaction rate is assessed by the non-hydrolyzed acetylcholine residue, which is determined by the amount of peracetic acid produced during the interaction with the excess of the hydrogen peroxide solution. The indicator reaction is the interaction of p-phenetidine with peracetic acid that leads to the formation of 4,4’-azoxyphenetole with λmax = 358 nm (log10 ε = 4.2).Results and discussion. As a result of the research conducted the linear dependence of the degree of inhibition of the enzymatic hydrolysis of acetylcholine (U, %) on the concentration of benzalkonium chloride was determined in the concentration range of (0.5 – 7.0) × 10–6 mol L-1 with the correlation coefficient of 0.999. The limit of quantitation was 1.9 × 10–6 mol L-1.Conclusions. As a result of the research conducted the kinetic enzymatic method for the quantitative determination of benzalkonium chloride has been developed by its inhibitory effect in the biochemical reaction of acetylcholine hydrolysis. This method is fast, cheap and easy to perform, does not require expensive equipment, and available for use in the field.

The inhibitory effect of xylan on enzymatic hydrolysis of cellulose is dependent on cellulose ultrastructure

Cellulose ◽  
2020 ◽  
Vol 27 (8) ◽  
pp. 4417-4428 ◽  
Author(s):  
Xindong Chen ◽  
Lian Xiong ◽  
Hailong Li ◽  
Liquan Zhang ◽  
Ge Yuan ◽  
...  
Keyword(s):  
Enzymatic Hydrolysis ◽  
Inhibitory Effect ◽  
Enzymatic Hydrolysis Of Cellulose ◽  
Hydrolysis Of Cellulose ◽  
Hydrolysis Of

scholarly journals SYNTHESIS OF GLYCOPROTEINS IN A SINGLE IDENTIFIED NEURON OF APLYSIA CALIFORNICA

1974 ◽  
Vol 61 (3) ◽  
pp. 649-664 ◽  
Author(s):  
Richard T. Ambron ◽  
James E. Goldman ◽  
Elizabeth Barnes Thompson ◽  
James H. Schwartz
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Mild Acid Hydrolysis ◽  
Giant Neuron ◽  
Electrophoretic Patterns ◽  
Identified Neuron ◽  
Hydrolysis Of ◽  
Mild Acid

Incorporation of L-[3H]fucose into glycoproteins was studied in R2, the giant neuron in the abdominal ganglion of Aplysia. [3H]fucose injected directly into the cell body of R2 was readily incorporated into glycoproteins which, as shown by autoradiography, were confined almost entirely to the injected neuron. Within 4 h after injection, 67% of the radioactivity in R2 had been incorporated into glycoproteins; at least 95% of these could be sedimented by centrifugation at 105,000 g, suggesting that they are associated with membranes. Extraction of the particulate fraction with sodium dodecyl sulfate (SDS), followed by gel filtration on Sephadex G-200 and polyacrylamide gel electrophoresis in SDS revealed the presence of only five major radioactive glycoprotein components which ranged in apparent molecular weight from 100,000 to 200,000 daltons. Similar results were obtained after intrasomatic injection of [3H]N-acetylgalactosamine. Mild acid hydrolysis of particulate fractions released all of the radioactivity in the form of fucose. When ganglia were incubated in the presence of [3H]fucose, radioactivity was preferentially incorporated into glial cells and connective tissue. In contrast to the relatively simple electrophoretic patterns obtained from cells injected with [3H]fucose, gel profiles of particulate fractions labeled with [14C]valine were much more complex.

Purification of isopenicillin N synthetase from Streptomyces clavuligerus

1986 ◽  
Vol 32 (12) ◽  
pp. 953-958 ◽  
Author(s):  
Susan E. Jensen ◽  
Brenda K. Leskiw ◽  
Leo. C. Vining ◽  
Yair Aharonowitz ◽  
Donald W. S. Westlake ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Streptomyces Clavuligerus ◽  
High Performance Liquid Chromatographic ◽  
Inhibitory Effect ◽  
Isopenicillin N Synthetase ◽  
Isopenicillin N

Isopenicillin N synthetase was purified from Streptomyces clavuligerus by sequential salt precipitation, ion-exchange and gel-filtration chromatography using both conventional open column and high-performance liquid chromatographic techniques. Material from the final purification step had a specific activity of 204.1 × 10−3 units/mg of protein which represented a 130-fold purification over the cell-free extract. The purified isopenicillin N synthetase was determined to have a molecular weight of 33 000 by sodium dodecyl sulfate – polyacrylamide gel electrophoresis and to have a Km of 0.32 mM with respect to its substrate δ-(L-α-aminoadipyl)-L-cysteinyl-D-valine. The enzyme showed a sensitivity to thiol-specific inhibitors with N-ethylmaleimide giving the strongest inhibitory effect.

Signal resistance of a soluble protein to enzymic proteolysis. An unorthodox approach to the isolation and purification of germin, a rare growth-related protein

1984 ◽  
Vol 62 (12) ◽  
pp. 1351-1353 ◽  
Author(s):  
Z. F. Grzelczak ◽  
B. G. Lane
Keyword(s):  
Soluble Protein ◽  
Sodium Dodecyl ◽  
Related Protein ◽  
Nucleosome Assembly ◽  
Polymeric Form ◽  
Isolation And Purification ◽  
Assembly Factor ◽  
Gastric Pepsin ◽  
Wheat Embryos ◽  
Hydrolysis Of

Onset of growth in germinating wheat embryos is marked by the conspicuous synthesis of germin, a soluble homopentameric protein. Germin is unusually stable in reducing environments containing sodium dodecyl sulfate, but the polymeric form is converted to a protomer (ca. 26 kdaltons) by brief heat treatment. In respect to these physical properties, germin is similar to nucleoplasmin, the putative nucleosome-assembly factor in Xenopus oocytes. To expand the comparison, we treated germin with gastric pepsin in the expectation that pepsin-catalyzed hydrolysis of germin might generate a series of fragments of the kind derived by pepsin digestion of nucleoplasmin. To our surprise, germin was refractory under conditions used to degrade nucleoplasmin. Further study has shown that germin exhibits a measure of stability toward the action of broad-specificity proteases which is unprecedented for a soluble protein. In this report, we document the remarkable resistance of this growth-related protein to enzymic proteolysis and project how this property may make it possible to isolate and purify an otherwise intractably rare, but "interesting" protein.

THE WHEAT LEAF PHOSPHATASES: VII. FURTHER STUDIES ON INHIBITORS AT pH 5.7

1963 ◽  
Vol 41 (1) ◽  
pp. 1727-1731 ◽  
Author(s):  
D. W. A. Roberts
Keyword(s):  
Acid Phosphatase ◽  
Enzymatic Hydrolysis ◽  
Inhibitory Effect ◽  
Substrate Specificities ◽  
Nitrophenyl Phosphate ◽  
Wheat Leaf ◽  
Hydrolysis Of

A survey of the inhibitory effect of various ribonucleosides, deoxyribonucleosides, sugars, phenols, and vitamins on the hydrolysis of 17 phosphatase substrates has been made. The more soluble ribonucleosides and deoxyribonucleosides inhibited the enzymatic hydrolysis of adenosine 5′-phosphate, phenolphthalein diphosphate, phenylphosphate, p-nitrophenyl phosphate, and in some cases the hydrolysis of adenosine 3′-phosphate, adenosine 2′-phosphate, and riboflavin 5′-phosphate. A 0.02 M concentration of orthophosphate inhibited the hydrolysis of all the compounds tested except adenosine 5′-phosphate and phenolphthalein diphosphate.These results, together with earlier findings, are discussed in terms of the concept that wheat leaf press juice contains two types of acid phosphatase, namely, β-glycerophosphatase and adenosine 5′-phosphatase. These two types of enzyme appear to have partially overlapping substrate specificities.

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