scholarly journals An Alternative Coupling Procedure for Preparing Activated Sepharose for Affinity Chromatography of Penicillinase

1976 ◽  
Vol 29 (4) ◽  
pp. 305 ◽  
Author(s):  
RG Coombe ◽  
AM George
Keyword(s):  
Affinity Chromatography ◽  
Room Temperature ◽  
Cyanogen Bromide ◽  
Dry Weight ◽  
Enzymic Activity ◽  
Covalent Attachment ◽  
Affinity Column ◽  
Coupling Procedure ◽  
Coupling Techniques ◽  
Cyanogen Bromide Activation

Most applications of affinity chromatography employ the cyanogen bromide activation scheme first devised by Axtm et al. (1967). Porath and Sundberg (1972) reported an alternative procedure in which phloroglucinol and divinylsulphone are used in activating reactions. The advantages of this scheme and parameters relevant to the activating reactions are reported here. Conditions for the attachment of various ligand molecules to sepharose using a divinylsulphone activation method are defined, and a comparison with cyanogen bromide activating and coupling techniques is drawn. a-Chymotrypsin is immobilized by covalent attachment to activated sepharose. The optimum coupling pH is 8� 0-8� 6 and the reaction is virtually complete after 20 h at room temperature. Conjugates containing as much as 2 g of enzyme per gram dry weight of polymer were obtained. The immobilized enzyme retained 41 % of the free enzymic activity. An affinity column of divinylsulphone-activated methicillin-sepharose was used to demonstrate the reversible adsorption of penicillinase.

scholarly journals A Facile and Efficient Bromination of Multi-Walled Carbon Nanotubes

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3161
Author(s):  
Sandra Zarska ◽  
Damian Kulawik ◽  
Volodymyr Pavlyuk ◽  
Piotr Tomasik ◽  
Alicja Bachmatiuk ◽  
...  
Keyword(s):  
Carbon Nanotubes ◽  
Room Temperature ◽  
Covalent Attachment ◽  
Closed Vessel ◽  
Dangling Bonds ◽  
Magnetic Stirrer ◽  
Defect Sites ◽  
Multi Walled Carbon Nanotubes ◽  
Walled Carbon Nanotubes ◽  
Covalently Bound

The bromination of multi-walled carbon nanotubes (MWCNT) was performed with vapor bromine in a closed vessel, and they were subjected to intensive stirring with a magnetic stirrer for up to 14 days. The efficiency of bromination was compared depending upon duration. The structure and surface of the crude and purified products were characterized by detailed physicochemical analyses, such as SEM/EDS, TEM, XRD, TGA, Raman, and XPS spectroscopies. The studies confirmed the presence of bromine covalently bound with nanotubes as well as the formation of inclusion MWCNT–Br2 complexes. It was confirmed that Br2 molecules are absorbed on the surface of nanotubes (forming the CNT-Br2 complex), while they can dissociate close to dangling bonds at CNT defect sites with the formation of covalent C−Br bonds. Thus, any covalent attachment of bromine to the graphitic surface achieved around room temperature is likely related to the defects in the MWCNTs. The best results, i.e., the highest amount of attached Br2, were obtained for brominated nanotubes brominated for 10 days, with the content of covalently bound bromine being 0.68 at% (by XPS).

PROPERTIES OF BOVINE Ac-GLOBULIN CONCENTRATES AND METHODS OF PREPARATION

1963 ◽  
Vol 41 (1) ◽  
pp. 2409-2421 ◽  
Author(s):  
Nobuo Aoki ◽  
Charles R. Harmison ◽  
Walter H. Seegers
Keyword(s):  
Human Plasma ◽  
Ammonium Sulphate ◽  
Protein Concentration ◽  
Room Temperature ◽  
Specific Activity ◽  
Barium Carbonate ◽  
Dry Weight ◽  
Physical Chemical ◽  
Bovine Plasma ◽  
Refrigerator Temperature

A procedure is described for retaining bovine plasma Ac-globulin activity as one part of the protein from plasma for every 1000 parts removed. The yields averaged 15%. The procedure involves removal of prothrombin with barium carbonate, isoelectric fractionation, fractionation with ammonium sulphate, chromatography on Amberlite IRC-50, and a second fractionation with ammonium sulphate. The procedure requires 2 days; however, the first day completes up to chromatography and the concentrate at that time is quite useful for many purposes. It is more stable than the product obtained after chromatography and the yields are higher. In absence of salts Ac-globulin is quite insoluble at pH 5.0. The final product usually contained some impurity. With the analytical ultra-centrifuge the S20in 0.1 M potassium chloride solution was found to be 4.2 at a protein concentration of 12.4 mg/ml. The specific activity was 1500 U./mg dry weight. Bovine plasma contains 120 U./ml or about 9 mg/100 ml. Assuming the same specific activity for human plasma the concentration is most likely near 1 mg/100 ml. The best stability conditions found were: 50% glycerol, pH 7.0, and 0.1 M calcium chloride. Under those conditions at room temperature all activity was retained 6 to 7 hours, at refrigerator temperature 24 hours, and at −60 °C for 1 month. In rabbits, antibodies were readily produced. Oxidizing agents destroyed the activity, while reducing agents did not, nor did they tend to stabilize. SH blocking agents destroyed the activity. The loss of activity in the presence of 0.0025 M parachloromercuribenzoate was recovered with 0.04 M cysteine. The molecule deteriorated while attempts were made to obtain physical chemical data; consequently, the molecular weight was calculated from an amino acid analysis and found to be 98,800. The reliability of this value is problematical. Human plasma was analyzed and found to contain 13 U./ml Ac-globulin. After 4 days storage, at room temperature, the prolonged prothrombin time of that plasma was completely restored with 13 units of Ac-globulin, which is equivalent to 8 μg.

scholarly journals Influence of Seed Hardening Techniques on Vigor, Growth and Yield of Wheat under Drought Conditions

2016 ◽  
Vol 4 (3) ◽  
pp. 121
Author(s):  
Amir Zaman Khan
Keyword(s):  
Grain Yield ◽  
Room Temperature ◽  
Dry Weight ◽  
Crop Productivity ◽  
Growth And Yield ◽  
Control Treatment ◽  
Sowing Seed ◽  
And Control ◽  
Peg 8000 ◽  
1000 Grain Weight

Exploring ways to improve stand establishment and crop productivity under abiotic stresses like drought is important. Two years experiments were conducted at University of Agriculture, Peshawar-Pakistan to examine the efficacy of six pre-sowing seed hardening agents. Seeds of wheat cultivar Uqab-2000 were hardened in six different chemicals of various concentration viz; PEG-8000 (10%), CaCl2 (4%), KNO3, (3%), Mannital (4%), NaCl (5%), Na2SO4 (2%) along with water soaking and dry seeds as control for 24 hours and drying back to original moisture content at room temperature. The soaking and drying of seeds was repeated twice for 12 hours. The results showed that pre-sowing hardening of seed with PEG-8000, CaCl2 and KNO3 gave higher germination, decreased days to 50% germination, increased shoot length, root length, seedling fresh and dry weight in laboratory experiment as compared with other hardening and control treatment. Under field conditions, maximum plant height (93.53cm), spikelet’s spike-1 (17.16), grains spike-1 (50.82), 1000 grain weight (39.97 g), grain yield (3482 kg ha-1) and maximum harvest index (32.5%) were observed in PEG-8000 hardened seed than control treatment (2872 kg ha-1). Seed hardened in PEG-8000, CaCl2 and KNO3 gave 30% increase in grain yield as compared to Mannital, NaCl and Na2SO4 which gave 15% increase in grain yield over control treatment.

Isolation Of Plasma Membrane Vesicles Of Human Platelet By Affinity Chromatography

1981 ◽  
Author(s):  
T Kobayashi ◽  
M Sakon ◽  
H Ohno ◽  
J Kambayshi ◽  
G Kösaki
Keyword(s):  
Plasma Membrane ◽  
Affinity Chromatography ◽  
Morphological Changes ◽  
Membrane Vesicles ◽  
Dry Weight ◽  
Appreciable Amount ◽  
Marker Enzyme ◽  
Plasma Membrane Vesicles ◽  
Platelet Suspension ◽  
Centrifugal Method

Platelets undergo a unique morphological changes leading to the formation of hemostatic plug. In recent years, its intermediaty metabolism has been extensively studied and the important function of plasma membrane in the platelet reaction has been recognized. The method of Barber and Jamieson has been employed in order to prepare plasma membrane vesicles of platelet of excellent quality but it is rather time consuming and the yield is relatively low. In this study, an attempt was made to isolate plasma membrane vesicles of human platelets by wheat germ agglutinin affinity chromatography.Freshly collected human citrated blood was subjected to glycerol loading and hypotonic lysis to obtain lysed platelet suspension. Then, it was applied to the affinity chromatography and the fraction of plasma membrane vesicles was eluted by 0.2 M N-acetyl glucosamine. Electron micrograph of the fraction showed round membrane vesicles with some scattered intracellular organelles. Several marker enzymes were assayed in the fraction. No appreciable amount of β-glucuronidase or cytochrome c oxidase was detected in the fraction, indicating no contamination of mitochondria or α-granules. Relatively high activity of G-6-Pase was detected, suggesting possible contamination of endoplasmic reticulum. The yield was 11.6% in dry weight and 7.9% in protein.By this method, the isolation was much faster than the centrifugal method and as low as 20 ml of human citrated whole blood may be used as starting material. Upon characterization of the plasma membrane fraction by electron microscopy and marker enzyme assays, the quality of the fraction was found comparable with the centrifugal method. The yield by this method was approximately two times higher than by the conventional method.

scholarly journals Affinity Chromatography: A Precise Method for Glycosylated Albumin Estimation

1985 ◽  
Vol 22 (1) ◽  
pp. 79-83 ◽  
Author(s):  
W G John ◽  
A E Jones
Keyword(s):  
Affinity Chromatography ◽  
Reference Range ◽  
Glycosylated Haemoglobin ◽  
Affinity Column ◽  
Albumin Concentration ◽  
Precise Method ◽  
Aminophenylboronic Acid

A method is described for the estimation, in plasma, of glycosylated albumin, using the commercially available affinity chromatographic material Glycogel B (immobilised m-aminophenylboronic acid on an agarose support) to separate the glycosylated and non-glycosylated albumin, followed by the analysis of albumin concentration using rocket Immunoelectrophoresis. Glycosylated albumin and glycosylated haemoglobin showed good correlation (r=0·91) when both are estimated by affinity chromatography. The glycosylated albumin method displays good within-batch (2·8–4·4% CV) and between-batch (2·9–5·4% CV) precision, and the method is not affected by haemolysis. Using this method a reference range of 2·0–5·4% was found for glycosylated albumin. Levels of glycosylated albumin in diabetics (10·06±3·23%) were found to be significantly higher ( P<0·001) than those in non-diabetics (3·72±0·85%). It was found that loading more than 3 mg of albumin on to a 1 mL affinity column must be avoided, as this appears to overload the column.

Purification by Affinity Chromatography of Glutathione Reductase (EC 1.6.4.2) from Escherichia coli and Characterization of such Enzyme

1984 ◽  
Vol 39 (9-10) ◽  
pp. 908-915 ◽  
Author(s):  
Anna M. Mata ◽  
M. Carmen ◽  
Juan López-Barea
Keyword(s):  
Escherichia Coli ◽  
Affinity Chromatography ◽  
Glutathione Reductase ◽  
Specific Activity ◽  
Total Yield ◽  
Coli Strain ◽  
Affinity Column ◽  
Heat Stable ◽  
Activity Loss ◽  
Fast Procedure

Abstract The glutathione reductase from Escherichia coli strain S33 was purified to homogeneity by a simple and fast procedure consisting of two affinity chromatography steps. After 40-80% ammonium sulfate fractionation, the enzyme was adsorbed to an N6-2′.5′-ADP-Sepharose affinity column from which it was specifically eluted by a 0 - 10 mᴍ NADP+ linear gradient. The enzyme was finally purified to homogeneity after a second affinity chromatography step in a C8-ATPR-Sepharose column, from which it was eluted by means of the same NADP+ gradient. Starting from 182 g of E. coli cells. 6.9 mg of pure enzyme was obtained after a 2632-fold purifi­cation, with a total yield of 63%. The pure enzyme showed a specific activity of 361 U/mg, and its absorption spectrum was characteristic of a flavoprotein. with an A272A450 of 7.84. The enzyme was a dimer with a molecular weight 109 000 and 40 Å hydrodynamic radius. The optimum pH were 7.5 and 4.5 with NADPH and NADH. respectively, as reductants. Apparent K′m values of 16, 377, and 66 μᴍ were determined at pH 7.5 for NADPH, NADH, and GSSG, respectively. Upon storage the enzyme was stable at pH values ranging from 7.5 to 9.5, being additionally stabilized by FAD. NADP+, dithiothreitol, or glycerol. The pure enzyme was quite heat stable, denaturing signifi­cantly only after 10 min at 70 0C. A marked activity loss was observed however, even at 0 °C, in the presence of 20 μᴍ NADPH. The enzyme was inactivated by low concentrations of para- hydroximercuribenzoate: the sensitivity towards such mercurial was greatly enhanced after reduction of the enzyme by NADPH.

Purification Of Antithrombin Ul Using Chinese Yellow ( Ocher Originated From China ) As A New Adsorbent And Heparin - Sepharose Affinity Chromatography

1981 ◽  
Author(s):  
H Suzuki ◽  
S Matsuo
Keyword(s):  
Affinity Chromatography ◽  
Room Temperature ◽  
Coagulation Factors ◽  
Centrifugal Separation ◽  
Thrombin Time ◽  
Blood Coagulation Factors ◽  
Α2 Macroglobulin ◽  
Sds Page ◽  
Adsorption Treatment ◽  
Immunologic Activity

In recent years, we have presented several reports with respect to Chinese Yellow ( ocher originated from China ) as a new adsorbent for blood coagulation factors. Adsorbability of this ocher is strong for many coagulation factors in plasma including fibrinogen, while quite weak for antithrombins. After ocher adsorption treatment, AT HI activity of plasma in comperison with the value before adsorption is 86 - 96 % in clotting assay ( Biggs thrombin time methods ) and 96 - 100 % in immunologic activity (detected by SRID ). In this study, purification of plasma AT HI, we have used this ocher as the adsorbent in pretreatment of plasma together with heparin - sepharose affinity chromatography. As the actual procedure, 5 g of ocher was added to 100 ml of ACD plasma, and the mixture was subjected to adsorption with stirring at room temperature to obtain the ocher adsorbed plasma, crude AT HI fraction, in the supernatant after centrifugal separation. Highly purified AT HI with a molecular weight of approximately 67,000 ( determined by SDS-PAGE ) was obtained from the crude AT HE fraction by means of heparin - sepharose affinity chromatography. The antiserum from rabbits immunized with the purified AT HI showed a single fuse precipitin line against the starting plasma, ocher adsorbed plasma and AT HI fraction. However, it did not exhibit reaction with α2-macroglobulin and α1-antitrypsin. From the above results, it has been found that the ocher is an effective adsorbent in pretreatment of plasma for AT HI purification.

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