A large-scale preparation and some physicochemical properties of recA protein.
Pure recA protein was easily obtained from Escherichia coli harboring plasmid pTM-2 which carried the recA gene by two chromatographic steps on phosphocellulose and DEAE-cellulose. RecA protein was stable in the pH range of 6 to 9 at 25 degrees C. RecA protein was found to aggregate highly under these conditions. Lowering of the protein concentration, the presence of glycerol, and lowering of the pH in the pH stability region diminished the extent of aggregation. The spectroscopic properties of recA protein were measured in the presence of 10% (v/v) glycerol. RecA protein had an absorption maximum at 278 nm. The value of a1% 1cm at 278 nm was determined to be 5.7. The tryptophyl fluorescence spectrum excited at 295 nm had an emission maximum at 340 nm and the quantum efficiency of recA protein relative to N-acetyl-L-tryptophanamide was determined to be 0.65. The CD spectrum of recA protein had negative double maxima at 210 and 220 nm. The alpha-helical content was estimated to be about 40% from the CD spectrum in the region of 200 to 250 nm. All three cysteinyl residues of recA protein were reacted with 5,5'-dithiobis(2-nitrobenzoic acid), and recA protein was found to have neither intramolecular nor intermolecular disulfide bond. The reactivities of the SH groups were changed by the presence of ATP or ADP. The denaturation of recA protein by guanidine hydrochloride was studied by measuring CD at 220 nm and tryptophyl fluorescence. The denaturation curve obtained by CD measurement consisted of two stages, one of which lies between 0 and 1.8 M and the other above 1.8 M guanidine hydrochloride. On the other hand, the denaturation curve obtained by fluorescence measurement consisted of a single transition in the concentration range of about 1 to 2.3 M guanidine hydrochloride.
D001426:Bacterial Proteins / Q000302:isolation & purification*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D003545:Cysteine / Q000378:metabolism; D004228:Dithionitrobenzoic Acid / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D046911:Macromolecular Substances; D011489:Protein Denaturation; D011938:Rec A Recombinases; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D046911:Macromolecular Substances; D004228:Dithionitrobenzoic Acid; D011938:Rec A Recombinases; D003545:Cysteine