Ribonucleotide reductase chromatography

Ribonucleotide reductase of E. coli consists of two non-identical subunits, which are purified together during the initial steps of the purification procedure however upon further purification the reductase separates into two subunits, proteins B1 and B2 (58). The overall yield of the two proteins is low, in particular that of protein Bl, which readily decomposes into smaller subunits. This decomposition can be counteracted by the addition of dithiols. An improved procedure for the purification of protein B1 using affinity chromatography on dATP-Sepharose has been described by Thelander (59). 

Unspecific inhibition of ribonucleotide reduction is produced by compounds like pyridoxal phosphate, or the sulfonated anthraquinone-triazine dye, Cibacron blue. They interact, like in many enzymes, with nucleotide binding domains where pyridoxal phosphate becomes covalently linked to lysine, or in that the dye occupies the whole nucleotide fold. The latter interaction permits its use in affinity chromatography of ribonucleotide reductases Likewise, EDTA is not a specific, nor a potent inhibitor, it may, for example, act by complexation of the structure-stabilizing Mg " ions in native holoenzymes. However the iron-promoted radical regeneration process appears far more susceptible to interference from EDTA ... 

E. coli differential centrifugation gel chromatography (M, = 2 x 10 ) DNA, phospholipids, ribonucleotide reductase, thioredoxin, thymidylate synthase, DNA polymerase 336... 

Purification of the steroid-active isoenzyme of alcohol dehydrogenase by affinity chromatography Isolation of ribonucleotide reductase by affinity chromatography Investigations of the effects of different spacer arms on the affinity chromatography of alkaline phosphatase Purification of P-450 haemeproteins of Rhizobium japonicum by affinity chromatography... 

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