Kazuo Okunuki

Preparation of subunits of flavocytochromes c derived from Chlorobium limicola f. thiosulfatophilum and Chromatium vinosum

Abstract The subunits of Chlorobium limicala f. thiosulfatophilum cytochrome c-553 and of Chromatium vinosum cytochrome c-552 have been obtained. Chlorobium cytochrome c-553 is split into the cytochrome and flavoprotein subunits by treatment with trichloroacetic acid; after the cytochrome is precipitated by 1–2% trichloroacetic acid, the cytochrome subunit is extractable with buffer, while the flavoprotein subunit is not dissolved. The subunits of Chromatium cytochrome c-552 can not be obtained by the trichloroacetic acid-treatment. The flavoprotein subunit of the Chromatium cytochrome is obtained by isoelectric focusing in the presence of 6 M urea and 1% mercaptoethanol, while the cytochrome subunit is prepared by gel filtration in the presence of 6 M urea with Sephacryl S-200. Molecular weights of the cytochrome and flavoprotein subunits from the Chlorobium cytochrome are 11,000 and 47,000, respectively, while those of the two subunits from the Chromatium cytochrome are 21,000 and 46,000, respectively. The molecule of each flavocytochrome c is composed of one molecule of each of the cytochrome and flavoprotein subunits.

ISOLATION OF BIOLOGICALLY ACTIVE PROTEINS

Publisher Summary This chapter summarizes many of the methods used to fractionate and to isolate proteins. Since the classical studies of Northrop and Kunitz and the school of E. J. Cohn, the Japanese investigators led by Akabori and Okunuki have been very successful in making use of both classical and modern techniques to isolate biologically active proteins in a highly purified form. The editors requested Professor Okunuki to write a detailed description of the methods used by his colleagues in the isolation of various cytochromes and bacterial enzymes. The chapter discusses cytochrome-α and its properties. It discusses cytochrome oxidase, cytochrome-b, and cytochrome-c. The chapter discusses the purification of Bakers yeast lactic dehydrogenase and bacterial α-amylase. Bacterial amylases can be classified at least into the following four groups: α -amylase and saccharifying amylase of Bacillus subtilis, amylase of Bacillus polymyxa, and amylase of Bacillus macerans. Of these amylases, α-amylase of Bacillus subtilis has been found industrially useful and it has been most widely investigated.