The Use of Disc Gel Electrophoresis with Nonionic Detergent in the Purification of Cytochrome f from Spinach Grana Membranes
Cytochrome f was purified from spinach grana membranes with a final yield of 42%. Using technology of disc gel electrophoresis with and without nonionic detergent, it was found that the cytochrome was intrinsically hydrophobic and existed as soluble aggregates at pH 8 which could be disaggregated by...
Saved in:
Published in: | The Journal of biological chemistry 1971-06, Vol.246 (11), p.3532-3541 |
---|---|
Main Authors: | , |
Format: | Article |
Language: | eng |
Subjects: | |
Online Access: | Get full text |
Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
Summary: | Cytochrome f was purified from spinach grana membranes with a final yield of 42%. Using technology of disc gel electrophoresis with and
without nonionic detergent, it was found that the cytochrome was intrinsically hydrophobic and existed as soluble aggregates
at pH 8 which could be disaggregated by 0.1% nonionic detergent to a single, stable, electrophoretic species. Disc gel electrophoresis
with non-ionic detergent was indispensable both in designing and in monitoring the purification procedure and served as a
final preparative chromatographic step. The cytochrome moved as a single electrophoretic species throughout the purification.
Cytochrome f was inactivated by urea, guanidine, and sodium dodecyl sulfate, which have commonly been used as disaggregating agents. Satisfactory
chromatography of cytochrome f in nonionic detergent solution was found possible in uncharged support media (disc gel electrophoresis or, alternatively,
on Sephadex G-200), but not on DEAE-cellulose.
Proof of homogeneity of cytochrome f is presented. The cytochrome appears to contain one heme-containing polypeptide chain and one non-heme chain, each about
31,000. The absorbance spectra of cytochrome f (reduced, oxidized, and reduced minus oxidized) at 22° and at -199° are presented. The cytochrome was not autoxidizable,
did not react with carbon monoxide, and contained no non-heme iron. The cytochrome was originally tightly bound, perhaps hydrophobically,
to grana membranes. |
---|---|
ISSN: | 0021-9258 1083-351X |