B. Carlton, C. Yanofsky
May 1, 1962
Citations
0
Influential Citations
19
Citations
Journal
The Journal of biological chemistry
Abstract
&faterials-The purified A protein used in these studies was obtained by a previously described method (2). The proteolytic enzymes were purchased from Worthington Biochemical Corporation. Carboxypeptidase A-DFPl (Lot 6064) had a proteolytic coefficient (C,) of 28 to 36 when assayed on 0.05 M carbobenzoxyglycyl-L-phenylalanine (3) at 37” by the photometric ninhydrin method of Moore and Stein (4). Carboxypeptidase B was treated with DFP and had a C1 of 67 to 81 as determined by the spectrophotometric assay of Folk et al. (5). Trypsin (Lot TR-706) and chymotrypsin (Lot CD. 578-81) were obtained as five times recrystallized and three times recrystallized preparations, respectively. Anhydrous hydrazinez was prepared by the method of Kusama (6). Hydruzinolysis-Samples of 0.19 pmole (5.7 mg) of purified A protein were dried over P20s and subjected to hydrazinolysis in sealed evacuated tubes by the method of Akabori et al. (7). The samples were incubated with 1.0 ml of anhydrous hydrazine at 105” for 5, 10, and 15 hours and the hydrazine was removed by evaporation in a vacuum desiccator over concentrated HzS04. The residue was then dried over PzOs for 4 hours and dissolved in 1.0 ml of distilled water, and 0.3 ml of isovaleraldehyde was added to precipitate the amino acid hydrazides. After standing at 0” for 30 minutes, the samples were centrifuged and the supernatant liquid was decanted. The precipitate was washed with 0.5 ml of water, and the wash was added to the first supernatant solution. Excess isovaleraldehyde was removed by extraction five times with 3 ml of redistilled ethyl acetate. The aqueous solution was adjusted to pH 2 with HCl and applied directly to the 150~cm column of a Spinco amino acid analyzer for the determination of free amino acids. Carboxypeptidase Digestion-Digestion of the A protein with