PAPAIN-PRODUCED FRAGMENTS OF HORSE ANTITOXIN: PHYS1COCHEMICAL AND IMMUNOLOGICAL PROPERTIES

1964 ◽  
Vol 42 (12) ◽  
pp. 1787-1792 ◽  
Author(s):  
J. J. Cohen ◽  
J. Myers ◽  
B. Rose ◽  
L. Goodfriend
Keyword(s):  
Ammonium Sulfate ◽  
Diphtheria Toxin ◽  
Unit Weight ◽  
Antibody Activity ◽  
Ammonium Sulfate Precipitation ◽  
Immunological Properties ◽  
Horse Antiserum

A mixture of γ- and β-globulins was prepared from horse antiserum to diphtheria toxin by ammonium sulfate precipitation. The mixture was treated with papain and separated into three fractions by chromatography on Sephadex G-100 columns. Peak 1 contained 5.0 S fragments; peak 2, 3.4 S; and peak 3, a mixture of 2.3 S and 0.3 S fragments. The 5.0 S component and the mixture of 2.3 S and 0.3 S components possessed divalent antibody activity, while the 3.4 S fragment was univalent. Although all fractions neutralized the dermonecrotic property of diphtheria toxin, the 5.0 S component neutralized 35 times as much toxin per unit weight as did the untreated globulin preparation.

PAPAIN-PRODUCED FRAGMENTS OF HORSE γ- AND β-GLOBULIN ANTITOXINS

1965 ◽  
Vol 43 (8) ◽  
pp. 1249-1255
Author(s):  
J. J. Cohen ◽  
J. Myers ◽  
B. Rose ◽  
L. Goodfriend
Keyword(s):  
Ammonium Sulfate ◽  
Diphtheria Toxin ◽  
Minor Components ◽  
Ammonium Sulfate Precipitation ◽  
Horse Antiserum

Preparations consisting mostly of γ- or β-globulin were obtained from a horse antiserum to diphtheria toxin by ammonium sulfate precipitation. These preparations were treated with papain and the digests separated on Sephadex G-100 columns. The γ-globulin digests were resolved into a major 3.4 S component and smaller minor components. The β-globulin digest contained similar fragments and, in addition, a major 5.0 S component. All fragments neutralized toxin, the most active being the β-globulin 5.0 S and 3.4 S fragments. The 3.4 S γ- and β-globulin fragments were univalent. The 5.0 S β-globulin fragment was divalent, and it could be reduced to 3.4 S subunits by treatment with 0.3 M 2-mercaptoethanol.

Extraction and Partial Purification of Protease from Bilimbi (Averrhoa bilimbi L.)

2013 ◽  
Vol 10 (2) ◽  
pp. 29
Author(s):  
Normah Ismail ◽  
Nur' Ain Mohamad Kharoe
Keyword(s):  
Molecular Weight ◽  
Protein Content ◽  
Ammonium Sulfate ◽  
Molecular Weight Distribution ◽  
Weight Distribution ◽  
Protease Activity ◽  
Protein Concentration ◽  
Temperature Stability ◽  
Partial Purification ◽  
Ammonium Sulfate Precipitation

Unripe and ripe bilimbi (Averrhoa bilimbi L.) were ground and the extracted juices were partially purified by ammonium sulfate precipitation at the concentrations of 40 and 60% (w/v). The collected proteases were analysed for pH, temperature stability, storage stability, molecular weight distribution, protein concentration and protein content. Protein content of bilimbi fruit was 0.89 g. Protease activity of both the unripe and ripe fruit were optimum at pH 4 and 40°C when the juice were purified at 40 and 60% ammonium sulfate precipitation. A decreased in protease activity was observed during the seven days of storage at 4°C. Molecular weight distribution indicated that the proteases protein bands fall between IO to 220 kDa. Protein bands were observed at 25, 50 and 160 kDa in both the unripe and ripe bilimbi proteases purified with 40% ammonium sulfate, however, the bands were more intense in those from unripe bilimbi. No protein bands were seen in proteases purified with 60% ammonium sulfate. Protein concentration was higher for proteases extracted with 40% ammonium sulfate at both ripening stages. Thus, purification using 40% ammonium sulfate precipitation could be a successful method to partially purify proteases from bilimbi especially from the unripe stage. 

scholarly journals A set of simple methods for detection and extraction of laminarinase

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ananthamurthy Koteshwara ◽  
Nancy V. Philip ◽  
Jesil Mathew Aranjani ◽  
Raghu Chandrashekhar Hariharapura ◽  
Subrahmanyam Volety Mallikarjuna
Keyword(s):  
Ammonium Sulfate ◽  
Ammonium Sulphate ◽  
Gold Standard ◽  
Direct Detection ◽  
Low Cost ◽  
Direct Analysis ◽  
Reducing Sugar ◽  
Ammonium Sulfate Precipitation ◽  
Simple Method ◽  
Functional Step

AbstractA carefully designed ammonium sulfate precipitation will simplify extraction of proteins and is considered to be a gold standard among various precipitation methods. Therefore, optimization of ammonium sulfate precipitation can be an important functional step in protein purification. The presence of high amounts of ammonium sulphate precludes direct detection of many enzymatically active proteins including reducing sugar assays (e.g. Nelson-Somogyi, Reissig and 3,5-dinitrosalicylic acid methods) for assessing carbohydrases (e.g. laminarinase (β (1–3)-glucanohydrolase), cellulases and chitinases). In this study, a simple method was developed using laminarin infused agarose plate for the direct analysis of the ammonium sulphate precipitates from Streptomyces rimosus AFM-1. The developed method is simple and convenient that can give accurate results even in presence of ammonium sulfate in the crude precipitates. Laminarin is a translucent substrate requiring the use of a stain to visualize the zones of hydrolysis in a plate assay. A very low-cost and locally available fluorescent optical fabric brightener Tinopal CBS-X has been used as a stain to detect the zones of hydrolysis. We also report simple methods to prepare colloidal chitin and cell free supernatant in this manuscript.

Preparation of Human Immunoglobulin by Ammonium Sulfate Precipitation

Vox Sanguinis ◽  
1976 ◽  
Vol 31 (6) ◽  
pp. 423-434
Author(s):  
A.F.S.A. Habeeb ◽  
Robert D. Francis
Keyword(s):  
Ammonium Sulfate ◽  
Human Immunoglobulin ◽  
Ammonium Sulfate Precipitation

scholarly journals Relationships between thrombopoiesis and erythropoiesis: with studies of the effects of preparations of thrombopoietin and erythropoietin

Blood ◽  
1976 ◽  
Vol 48 (4) ◽  
pp. 547-558 ◽  
Author(s):  
BL Evatt ◽  
JL Spivak ◽  
J Levin
Keyword(s):  
Ammonium Sulfate ◽  
Red Cells ◽  
Red Cell ◽  
Erythropoietic Activity ◽  
Rabbit Plasma ◽  
Ammonium Sulfate Precipitation ◽  
Sequential Fractionation ◽  
Platelet Production ◽  
Cell Production

Abstract The effects of administration of partially purified human urinary erythropoietin and rabbit thrombopoietin, and of endogenously produced erythropoietin and thrombopoietin on both red cell and platelet production were examined in mice. Partially purified thrombopoietin was prepared from rabbit plasma by sequential fractionation with ammonium sulfate precipitation, and DEAE and Sephadex G-100 chromatography. Preparations of thrombopoietin and partially purified human urinary erythropoietin (NIH No. H-11-TaLSL) were administered subcutaneously to normal mice, and the rate of incorporation of selenomethionine-75 Se into platelets was measured as an index of thrombopoietic activity of the infused material. Erythropoietin and thrombopoietin were assayed for erythropoietic activity by measuring the rate of appearance of 59Fe in the red cells of posthypoxic polycythemic mice. Preparations containing thrombopoietin had barely measurable erythropoietic activity, and 7 units of partially purified erythropoietin had little thrombopoietic activity. When endogenous levels of erythropoietin were increased by hypoxia, platelet production was not enhanced. Similarly, increased levels of thrombopoietin, induced in response to thrombocytopenia produced by platelet antiserum, did not alter red cell production. These data suggest that physiologically increased levels of thrombopoietin do not stimulate erythropoiesis, and that physiologically increased levels of erythropoietn do not stimulate thrombopoiesis. However, currently available, partially purified preparations of erythropoietin and thrombopoietin may be capable of stimulating both platelet and red cell production if used in sufficient quantities.

Purification and kinetic studies of the hemolysin from Escherichia coli

1971 ◽  
Vol 17 (6) ◽  
pp. 741-745 ◽  
Author(s):  
Peter Zwadyk Jr. ◽  
I. S. Snyder
Keyword(s):  
Escherichia Coli ◽  
Ammonium Sulfate ◽  
Reaction Rate ◽  
Kinetic Studies ◽  
Ammonium Sulfate Precipitation ◽  
Complex Curve ◽  
Sheep Erythrocytes

The hemolysin of Escherichia coli has been concentrated 23-fold by ethanol and ammonium sulfate precipitation. Further evidence has been presented that the hemolysin is a protein or peptide. Kinetic studies of the hemolytic reaction reveal a complex curve which consists of a lag, an area of accelerated reaction rate, and a plateau. Kinetic studies also show that the hemolytic reaction can be best demonstrated at a pH of 7.9 and a temperature of 30 °C using a 1% suspension of sheep erythrocytes.

Purification and properties of a β-hexosidase from Sporobolomyces singularis

1969 ◽  
Vol 47 (11) ◽  
pp. 1021-1025 ◽  
Author(s):  
J. A. Blakely ◽  
S. L. MacKenzie
Keyword(s):  
Molecular Weight ◽  
Ammonium Sulfate ◽  
Sedimentation Coefficient ◽  
Polyacrylamide Gel ◽  
Ammonium Sulfate Precipitation ◽  
Purification And Properties

A β-hexosidase has been isolated from Sporobolomyces singularis by conventional techniques involving ammonium sulfate precipitation and chromatography on columns of Sephadex G-200 and DEAE-Sephadex A-50. Electrophoresis on polyacrylamide gel was used as the final preparative step. The sedimentation coefficient (s°20,w) of the enzyme was 7.6 and its molecular weight was in the range 140 000–145 000. Although the β-hexosidase performed the functions of a β-D-galactoside galactohydrolase (β-galactosidase), it also catalyzed the hydrolytic function normally performed by a β-D-glucoside glucohydrolase; both these functions appear to reside in the same molecule.

scholarly journals The membrane attack mechanism of complement: the three polypeptide chain structure of the eigth component (C8).

1976 ◽  
Vol 143 (5) ◽  
pp. 1131-1139 ◽  
Author(s):  
W P Klob ◽  
H J Müller-Eberhard
Keyword(s):  
Ion Exchange ◽  
Human Serum ◽  
Ammonium Sulfate ◽  
Gel Electrophoresis ◽  
Polypeptide Chain ◽  
Polyacrylamide Gel Electrophoresis ◽  
Chain Structure ◽  
Ammonium Sulfate Precipitation ◽  
Ion Exchange Column ◽  
Polypeptide Chains

The purification of human C8 in milligram quantities from outdated human serum was achieved by ammonium sulfate precipitation (37.5-50% saturation) and ion exchange column chromatography employing CM-32 cellulose and QAE-Sephadex. The yield of C8 activity ranged from 2-9%, and the average purification was 1,700-fold. Fully reduced C8 was shown by SDS polyacrylamide gel electrophoresis to have three polypeptide chains which were present in equimolor ratios: alpha, 77,000 daltons; beta, 63,000 daltons; and gamma, 13,700 daltons. C8 denaturation by SDS and urea in the absence of reducing agents revealed two noncovalently linked subunits: alpha-gamma, 99,000 daltons, and beta, 75,000 daltons.

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