PMID: 6983889Nov 23, 1982Paper

Isolation of bacterial luciferases by affinity chromatography on 2,2-diphenylpropylamine-Sepharose: phosphate-mediated binding to an immobilized substrate analogue

Biochemistry
T F Holzman, T O Baldwin

Abstract

A covalently immobilized form of an inhibitor of bacterial luciferase, 2,2-diphenylpropylamine (D phi PA), was an effective affinity resin for purifying this enzyme from several distinct bacterial species. The inhibitor is competitive with the luciferase aldehyde substrate but enhances binding of the flavin substrate FMNH2 (reduced riboflavin 5'-phosphate); comparable binding interactions occur with luciferase, the immobilized inhibitor D phi PA-Sepharose, and the substrates [Holzman, T. F., & Baldwin, T. O. (1981) Biochemistry 20, 5524-5528]. The effect of FMNH2 on the binding of luciferase to D phi PA-Sepharose was mimicked by inorganic phosphate; the luciferase-phosphate complex had a greater affinity for D phi PA-Sepharose than did luciferase. This observation led to the development of a method using D phi PA-Sepharose to purify bacterial luciferase. When crude enzyme in a high-phosphate buffer was applied to a column of the affinity matrix, the luciferase activity was removed from solution. After the column was washed with the same buffer to remove unbound protein, the luciferase was eluted with a non-phosphate cationic buffer. The affinity column has proven useful for rapid purification of luciferase in much greater yield...Continue Reading

References

Jan 1, 1977·Annual Review of Microbiology·J W Hastings, K H Nealson
Jul 1, 1978·Analytical Biochemistry·P T Matsudaira, D R Burgess
Apr 22, 1977·Science·E G Ruby, K H Nealson
Apr 23, 1974·Biochemical and Biophysical Research Communications·C A WatersJ W Hastings

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Citations

Apr 1, 1991·Journal of Bioluminescence and Chemiluminescence·J C Makemson, J W Hastings
Dec 1, 1987·Proceedings of the National Academy of Sciences of the United States of America·S C DaubnerT O Baldwin
Nov 15, 1993·Proceedings of the National Academy of Sciences of the United States of America·G C FlynnF W Dahlquist
Feb 1, 1992·Photochemistry and Photobiology·H KarataniJ W Hastings
Aug 1, 1993·Trends in Biochemical Sciences·C LindbladhL Bülow
Jul 15, 1987·Biochemical and Biophysical Research Communications·P MacherouxJ W Hastings
Jan 5, 1982·Journal of Molecular Biology·B Sedgwick, T Lindahl
May 1, 1992·Archives of Biochemistry and Biophysics·J C MakemsonJ M Quirke
Jul 1, 1989·Journal of Bioluminescence and Chemiluminescence·T O BaldwinM M Ziegler
Oct 1, 1989·Applied and Environmental Microbiology·P ColepicoloJ W Hastings

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