Abstract
The lysozyme of bacteriophage T4 was purified to apparent homogeneity from lysates of the phage grown on Escherichia coli. The enzyme is a single polypeptide chain of molecular weight 19,000, with a single NH2-terminal methionine residue and a single COOH-terminal leucine residue. The amino acid composition of the protein was determined. The phage lysozyme exhibits a much greater specific activity when assayed with E. coli as a substrate than does egg white lysozyme. The enzyme was found to have muramidase activity, as egg white lysozyme does, when cell walls of Micrococcus lysodeikticus were used as substrate. The stability of the enzyme and the pH of optimum activity are described.
Highlights
A standard curve was constructed by measuring the absorbance given by a series of different concentrations of phage lysozyme
The carboxyl-terminal amino acid was investigated by the hydrazinolysis procedure [16]) the released carboxyl-terminal amino acid being identified by amino acid analysis after treatment with aldehyde [17]
Ten-liter batches of Fraser medium in 25-l&r culture bottles were inoculated with 400 ml of an overnight culture of E. coli B grown in the same medium and were incubated with shaking at 37”
Summary
The lysozyme of bacteriophageT4 was purified to apparent homogeneity from lysates of the phage grown on Escherichia coli. Lysozyme of wild type T4 phage has been purified and characterized with respect to activity, stability, amino acid composition, and terminal amino acids. Assay 1: A fresh suspension (3 ml) of 50 mg of lyophilized E. coli cells in 100 ml of 0.05 M Tris-HCl buffer, pH 7.4, was mixed with 0.2 ml of the enzyme sample in a cuvette in a spec-. The cell suspension (0.9 ml) was incubated with 0.1 ml of the enzyme sample for 5 min at 37”, and the absorbance at 350 rnp was measured. A standard curve was constructed by measuring the absorbance given by a series of different concentrations of phage lysozyme. Amino Acid Analysis-Free ammonia was removed by incubating 5-mg amounts of samples to be analyzed in 0.5 ml of 0.2 M borate buffer, pH 9.0, in a boiling water bath for 5 min. The carboxyl-terminal amino acid was investigated by the hydrazinolysis procedure [16]) the released carboxyl-terminal amino acid being identified by amino acid analysis after treatment with aldehyde [17]
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