Joseph J. Kolb

On the component proteins of calf thymus nucleoprotein.

Abstract By a study of experimental variables, a standardized procedure for the preparation of calf thymus nucleoprotein of essentially constant phosphorus and sulfur content has been developed. The nucleic acid-free protein has been isolated quantitatively by a modified Sevag-Mirsky procedure. The protein has been quantitatively separated into histone and residual protein by treatment with HCl at pH 2.5. The histone precipitated as a single substance, but on ultracentrifugation showed two components. These have been separated and partly characterized. One (“fast”), representing 63% of the histone and yielding molecular weight estimates of 130,000–140,000, contains 0.23% tryptophan, 0.67% cystine, and 2.4% methionine. The other component (37%, “slow”), of estimated molecular weight of 11,000–16,000, contains0.82% methionine, and is free of tryptophan and cystine.

Microdetermination of lipid phosphorus as a measure of bacterial membrane substance.

Abstract A microprocedure for the precise determination of bacterial lipid phosphorus under conditions where it represents less than 5% of the total phosphorus, is presented, together with analytical results which indicate that bacterial lipid phosphorus may serve as a quantitative index of bacterial membrane substance.

Carbohydrate analysis of bacterial substances by a new anthrone procedure

Abstract A micro procedure is presented for the application of the anthrone reaction to the quantitative characterization of the carbohydrates of bacteria and their anatomical parts. The data obtained indicate that the principal saccharide components of Streptococcus faecalis are glucose and rhamnose, and that the cell membrane contains glucose but no rhamnose, while rhamnose and glucose are both present in the cell wall and both absent in the cytoplasm of exponentially growing cells.

Bacterial nucleate and phosphorus partition

Methods for the measurement of nucleates and other phosphorus-containing classes in bacteria have been studied. Conditions are presented for the optimal extraction of low- and high-molecular nucleates as measured by ultraviolet absorbancy. Various cell crops were fractionated by a combination of hot and cold extractions and mechanical separations, and characterized by phosphorus and absorbancy determinations. Eight phosphorus fractions can be distinguished. They are (expressed as per cent of the total phosphorus and presented in terms of approximate ranges encountered in Streptococcus faecalis crops of different nutritional origin): inorganic or easily hydrolyzable P (10–26), lipid P (3–10), presumptive wall teichoate P (5–11), soluble nucleoside monophosphate P (4–9), other soluble P (polyphosphates, phosphorylated intermediates, teichoates, etc.) (0–40) DNA P (4–8), RNA P (17–67), and undefined nonextractable P (3–14). Cold and hot extractions with sulfuric acid were found to give results substantially identical with those obtained by perchloric acid of the same normalities. The difficulties that attend determination of nitrogen in perchloric extracts are absent in sulfuric extracts. The study which included analysis of commercial RNA and DNA preparations, indicates that the molar absorbancy of nucleate phosphorus at 260 mμ differs substantially among different preparations, presumably because of differences in their nucleobase ratios. For dependable determination of total nucleates, phosphorus partition is recommended. Application of the diphenylamine reaction to the determination of DNA in bacterial products, and in growing bacterial cultures, has also been studied, and procedures have been suggested.