Purification and characterization of a catalase from photosynthetic bacterium Rhodospirillum rubrum S1 grown under anaerobic conditions.
The photosynthetic bacterium, Rhodospirillum rubrum S1, when grown under anaerobic conditions, generated three different types of catalases. In this study, we purified and characterized the highest molecular weight catalase from the three catalases. The total specific catalase activity of the crude cell extracts was 88 U/mg. After the completion of the final purification step, the specific activity of the purified catalase was 1,256 U/mg. The purified catalase evidenced an estimated molecular mass of 318 kDa, consisting of four identical subunits, each of 79 kDa. The purified enzyme exhibited an apparent Km value of 30.4 mM and a Vmax of 2,564 U against hydrogen peroxide. The enzyme also exhibited a broad optimal pH (5.0-9.0), and remained stable over a broad temperature range (20 degrees C-60 degrees C). It maintained 90% activity against organic solvents (ethanol/chloroform) known hydroperoxidase inhibitors, and exhibited no detectable peroxidase activity. The catalase activity of the purified enzyme was reduced to 19% of full activity as the result of the administration of 10 mM 3-amino-1,2,4-triazole, a heme-containing catalase inhibitor. Sodium cyanide, sodium azide, and hydroxylamine, all of which are known heme protein inhibitors, inhibited catalase activity by 50% at concentrations of 11.5 microM, 0.52 microM, and 0.11 microM, respectively. In accordance with these findings, the enzyme was identified as a type of monofunctional catalase.
Kang
Yoon-Suk
YS
Department of Life Science, College of Natural Science, Cheju National University, Jeju 690-756, Republic of Korea.
Lee
Dong-Heon
DH
Yoon
Byoung-Jun
BJ
Oh
Duck-Chul
DC
D000640:Amitrole / Q000494:pharmacology; D000693:Anaerobiosis; D002374:Catalase / Q000737:chemistry* / Q000187:drug effects / Q000302:isolation & purification; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004795:Enzyme Stability; D006420:Hemeproteins / Q000037:antagonists & inhibitors; D006861:Hydrogen Peroxide / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D008670:Metals / Q000494:pharmacology; D008970:Molecular Weight; D010788:Photosynthesis; D012247:Rhodospirillum rubrum / Q000201:enzymology*; D013696:Temperature
D004791:Enzyme Inhibitors; D006420:Hemeproteins; D008670:Metals; D006861:Hydrogen Peroxide; D002374:Catalase; D000640:Amitrole