Quantitative Affinity Chromatographic Studies of Mitochondrial Cytochrome c Binding to Bacterial Photosynthetic Reaction Center, Reconstituted in Liposome Membranes and Immobilized by Detergent Dialysis and Avidin–Biotin Binding
✍ Scribed by Qing Yang; Xue-Ying Liu; Masayuki Hara; Per Lundahl; Jun Miyake
- Publisher
- Elsevier Science
- Year
- 2000
- Tongue
- English
- Weight
- 101 KB
- Volume
- 280
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
In order to study the affinity binding of c-type cytochromes to the photosynthetic reaction center (RC) by quantitative affinity chromatography (QAC), RC from Rhodobacter sphaeroides was reconstituted into liposomes composed of egg phosphatidylcholine (EPC) and 2 mol% of biotinyl phosphatidylethanolamine simultaneously as the liposomes were formed and immobilized in (strept)avidin-coupled gel beads by rotary detergent dialysis. The immobilized amount was up to 80 nmol of RC and 33 mol of lipid/g of moist gel in streptavidin-coupled Sephacryl S-1000 gel. By QAC frontal runs, retardation of mitochondrial cyt c on immobilized RC liposome columns was demonstrated. The dissociation constant for the RC-cyt c interaction was determined to be 0.20 -0.57 M. QAC studies also allowed evaluation of the orientation of reconstituted RC in immobilized liposomes by comparison of the total amount of cyt c binding sites with the amount of available binding sites obtained by QAC. It seems that the RC proteoliposomes immobilized in Sephacryl S-1000 gel exposed the cyt c binding sites on the outer surface of the liposomes due to effects of the gel network pore size and the resulting liposomal size.