SOME PROPERTIES OF A BACILLOMYCIN b-CYTOCHROME c COMPLEX (1951)

Interactions between proteins and ions in solution, and between different protein molecules, may follow adjustment of the electrochemical environment to produce net charges of opposite sign.The isolated protein, cytochrome c, is isoelectric in the alkaline range owing to the basic group in the porphyrin part, of the molecule (1) and probably to the unusually high content of the strongly cationic lysine in the protein moiety (2).Thus, this protein represents a potential cation for complex formation with a large variety of the generally more acid proteins and other smaller anions over a wide pH range.In this respect, the system is analogous to the cationic behavior of protamines and histones in interactions with hemoglobin and casein (3), with insulin (4), and with toxoids (5, 6).Recently, Klotz et al. ( 7) have investigated the binding of penicillins by proteins through differential-dialysis.Spectroscopic evidence is given for the inter-BACILLOMYCIN b-CYTOCHROME C COMPLEX tions of the present study were determined spectrophotometrically, with constants derived earlier (11) .2Bacillomycin b-The antifungal principle derived from cultures of a B. subtilis strain was isolated by solvent extraction of the precipitate from acidified broth, as described by Landy et al. (8).Purification was subsequently effected by repeated crystallizations from methanol-water mixtures at acid PH.~The purified preparation occurs as colorless microscopic need,les or rosettes.Elementary analysis reveals carbon, hydrogen, and nitrogen, but no sulfur, phosphorus, or halogens.No significant optical rotation in alcohol solution was observed.The crystals are readily soluble in concentrated acids and lower alcohols and insoluble in ether, hydrocarbons, and dilute acids below pH 4.0 to 4.5.Water solubility is slow at neutral pH and relatively rapid in alkaline buffers.Bacillomycin b is apparently a polypeptide possessing a titratable acid function.Qualitative analyses for amino acids upon residues, following hydrolysis with 6 N HCl in sealed tubes at 100" for 24 hours, by two-dimensional paper chromatography (12), indicated significant quantities of glutamic and aspartic acids, tyrosine, serine, and threonine, and possible traces of alanine, valine, and isoleucine in some preparations.The presence of the last three, however, was not confirmed in the most purified samples by microbiological tests (13).In these procedures Streptococcus jaecalis R and Leuconostoc mesenteroides were employed as test organisms.Whereas the chromatogram was indicative of a high relative concentration of glutamic acid, the microbiological procedure indicated only a trace effect upon the growth of the test organisms.This suggested the possibility of a relatively high n-glutamic acid content, a condition consistent with the findings of Ivanovics and Bruckner in some of the metabolites of organisms in the Bacillus subtilis-mesentericus group ( 14) and with the occurrence of the same configuration in some of the amino acids elaborated by a related aerobic spore-forming organism, Bacillus brevis (15).A high n-amino acid content in bacillomycin b was subsequently determined by the enzymatic action of the specific n-amino acid oxidase of Krebs ( 16) upon the hydrolysis products.This reaction was measured by oxygen uptake in direct Warburg manometers and was confirmed by the calorimetric test of Sealock ( 17), in which the a-keto acid formed in the enzyme reaction is determined with 2,4-dinitrophenylhydrazine.By * The relationship between the molecular extinction coefficient (e,) and the optical density of the solution (D) is expressed as cm = D/(c X d) (c, concentration (moles per liter), d, length of optical path in solution (1 cm.)).The concentration of cytochrome c in solution may then be calculated as c (mg. per ml.) = (D X 12,270 (molecular weight))/27,840 (cm) = D X 0.441.3 Alburn, H. E., and Williams, E. C., unpublished data.