DOUBLE TRITON X-114 PHASE PARTITIONING FOR THE PURIFICATION OF PLANT CYTOCHROMES P450 AND REMOVAL OF GREEN PIGMENTS

A double Triton X-114 phase partitioning procedure that separates plan t cytochromes P450 from green pigments and provides an extract highly enriched in total cytochromes P450 has been developed. Upon phase part itioning in Triton X-114, plant cytochromes P450 have previously been found to partition to the pigmented detergent rich phase. These partit ionings were carried out using phosphate buffer. We found that the par titioning of the cytochromes P450 could be shifted to a pigment-free T riton X-114 poor phase by changing the buffer component to berate. The protein extract containing the cytochromes P450 but devoid of green p igment was subjected to a second phase partitioning step before which the buffer was changed from berate to phosphate. This second phase par titioning step produced a Triton X-114-rich phase highly enriched in c ytochromes P450 proteins compared to the microsomal starting material as monitored by sodium dodecyl sulfate-polyacrylamide gel electrophore sis, cytochrome P450 reconstitution assays, and Western blotting. The yield of the double phase partitioning purification procedure is about 26% which is high compared to the yields obtained at similar stages o f purification using column chromatography. The double phase partition ing procedure takes 3-4 h to complete. This is very fast compared to t raditional purification schemes for cytochromes P450 which involve mul tiple of column chromatographic steps. Plant cytochromes P450 are labi le, low abundant proteins that are difficult to isolate. The double Tr iton X-114 phase partitioning here reported thus constitutes a versati le, efficient purification procedure circumventing many of the problem s previously encountered. (C) 1998 Academic Press.