scholarly journals Ion-exchange chromatography in the presence of the non-ionic dissociating agent chloral hydrate. Application to a membrane protein, bovine heart cytochrome c oxidase

1982 ◽  
Vol 201 (1) ◽  
pp. 227-231 ◽  
Author(s):  
D C Griffin ◽  
M Landon
Keyword(s):  
Cytochrome C ◽  
Ion Exchange ◽  
Cytochrome C Oxidase ◽  
Chloral Hydrate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Exchange Chromatography ◽  
Human Haemoglobin ◽  
Bovine Heart ◽  
Model Protein ◽  
Restricted Use

In previous work we have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) is a potent non-ionic protein dissociating agent. We have employed it in systems of polyacrylamide-gel electrophoresis and have demonstrated the presence of 15 components in a preparation of bovine heart cytochrome c oxidase [Griffin & Landon (1981) Biochem. J. 197, 333-344]. Here we describe the use of solutions containing aq. 100% (w/v) chloral hydrate in the ion-exchange column chromatographic separation on CM-cellulose of the alpha- and beta-chains of human haemoglobin, which we have employed as a model protein of known structure. We also describe the use of similar procedures in order to fractionate the polypeptide components of bovine heart cytochrome c oxidase. An effective separation has been obtained and we suggest that chloral hydrate-containing solutions could have general application in the ion-exchange-chromatographic analysis of membrane proteins, a procedure that has had restricted use owing to the inadequacy of non-ionic dissociating agents available previously.

scholarly journals Additional components of bovine heart cytochrome c oxidase demonstrated by high-resolution polyacrylamide-gel electrophoresis in the presence of chloral hydrate

1981 ◽  
Vol 197 (2) ◽  
pp. 333-344 ◽  
Author(s):  
D C Griffin ◽  
M Landon
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Gel Electrophoresis ◽  
Chloral Hydrate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Bovine Heart ◽  
Electrophoretic Separations ◽  
New Procedures

We have shown that aq. 100% (w/v) chloral hydrate (2,2,2-trichloroethane-1,1-diol) dissociates bovine heart cytochrome c oxidase. We have developed new procedures of polyacrylamide-gel electrophoresis in the presence of chloral hydrate that permit variation in the pH of the separation, and, by using these procedures, we have observed 15 components in preparations of the enzyme. This number contrasts with the eight bands that were seen on electrophoresis in the presence of SDS (sodium dodecyl sulphate) and urea. We have isolated material from these eight bands and have characterized each by electrophoresis in the presence of chloral hydrate. Twelve of the fifteen components that were seen by electrophoresis in chloral hydrate were identified as constituents of the eight bands seen by electrophoresis in the presence of SDS and urea. Two-dimensional electrophoretic separations confirmed these identifications ans showed that the other three components which were resolved as discrete bands by electrophoresis in the presence of chloral hydrate appeared to be diffusely present in the electrophoretic separations performed in the presence of SDS and urea, which suggested anomalous behaviour in that detergent. Trypsin treatment of cytochrome c oxidase caused total loss, as observed by electrophoretic separations in the presence of chloral hydrate, of a number of components. The trypsin-sensitive components included all of those that behaved anomalously in the presence of SDS and urea. Chloral hydrate is a potent non-ionic dissociating agent for cytochrome c oxidase and its use in polyacrylamide-gel electrophoresis, with variation in the pH of the gel, permits charge-dependent separations that should have general application in the analysis of membrane proteins.

scholarly journals A new procedure for the purification of monodisperse highly active cytochrome c oxidase from bovine heart

1987 ◽  
Vol 242 (2) ◽  
pp. 417-423 ◽  
Author(s):  
Y Li ◽  
A Naqui ◽  
T G Frey ◽  
B Chance
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Gradient Centrifugation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Gel Filtration ◽  
Homogeneous State ◽  
Bovine Heart ◽  
Highly Active ◽  
Solubilized Enzyme

A simple and rapid method for the isolation of a large quantity of cytochrome c oxidase from bovine heart mitochondria was developed, based on selective solubilization of mitochondrial protein with first Triton and then lauryl maltoside. Gel filtration shows that the lauryl maltoside-solubilized oxidase preparation is in a hydrodynamically homogeneous state with a Stokes radius of 7.5 +/- 0.2 nm. It contains 8.0 mumol of haem (with an a/a3 ratio of 1)/g of protein. The catalytic constant (maximum turnover number) with respect to cytochrome c approaches 600 S-1. After further purification of the solubilized enzyme on a sucrose-gradient centrifugation, the purified enzyme has a haem content of 10.3 mumol/g of protein and eight major polypeptide bands shown on SDS/polyacrylamide-gel electrophoresis.

scholarly journals Isolation, partial purification and characterization of phospholipase A2 from Naja Katiensis venom

2021 ◽  
Vol 13 (2) ◽  
pp. 107-112
Author(s):  
C.F. Okechukwu ◽  
P.L. Shamsudeen ◽  
R.K. Bala ◽  
B.G. Kurfi ◽  
A.M. Abdulazeez
Keyword(s):  
Ion Exchange ◽  
Phospholipase A2 ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Crude Venom

The most effective and acceptable therapy for snakebite victims is the immediate administration of antivenin which is limited by problems of hypersensitivity reactions in some individuals and its inability to resolve the local effects of the venom. The aim of this study was to isolate, partially purify and characterize phospholipase A2 from Naja Katiensis venom. Phospholipase A2 was partially purified via a two-step process: gel filtration on Sephadex G-75 and ion exchange chromatography using CM Sephadex, and subjected to SDS-PAGE analysis. From the results, the specific activity of the partially purified PLA2 decreased from 0.67μmol/min/mg in crude venom to 0.29μmol/min/mg after ion exchange chromatography with a yield of 5% and purification fold of 0.43. The optimum temperature of the purified PLA2 was found to be 35ºC and optimum p.H of 7. velocity studies for the determination of kinetic constants using L-a-lecithin as substrate revealed a Km  of 1.47mg/ml and Vmax  of 3.32μ moles/min/mg. The sodium dodecyl sulphate polyacrylamide gel electrophoresis of the purified PLA2 showed a distinct band with molecular weight estimated to be 14KDa. In conclusion, the present study shows that phospholipase A2 was isolated, purified and characterized. This may serve as a promising candidate for future development of a novel anti-venin drug.

The isolation of bovine-heart cytochrome c oxidase subunits Dependence on phospholipid and cholate content

1979 ◽  
Vol 548 (2) ◽  
pp. 397-416 ◽  
Author(s):  
F.E.A.M. Verheul ◽  
J.C.P. Boonman ◽  
J.W. Draijer ◽  
A.O. Muijsers ◽  
D. Borden ◽  
...  
Keyword(s):  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Bovine Heart
Sign in / Sign up

Export Citation Format

Share Document