The qualitative and quantitative amino acid content of bacitracin A.

The hydrolysis of bacitracin has been under study in this laboratory at different times and with different preparations during the past few years. A report of the experiments has been withheld pending more conclusive evidence that the material to be used for the hydrolysis was a single substance in the chemical sense. The results reported in two accompanying papers dealing with fractionation of bacitracin (1) and molecular weight determination by partial substitution (2) now appear to be sufficiently clear cut to justify a more complete report on the hydrolysis experiments. As reported in an earlier communication (3), bacitracin appears to contain peptide linkages which are strongly resistant to complete hydrolysis. Moreover, colored humin-like material is formed upon hydrolysis, the amount depending on the concentration of the peptide initially used. A solution of 1 gm. of peptide in 200 ml. of G N HCl appeared to give the best result. In earlier studies on less highly purified material, refluxing for 42 hours gave lower recoveries by starch chromatography’ for most of the amino acids but a considerably higher recovery of ammonia than was obtained when the duration of hydrolysis was only 18 hours. This indicated partial destruction of certain of the amino acids by some hydrolytic product of the peptide. A 24 hour hydrolysis period at 110” in a sealed evacuated tube was finally selected for careful study, however, with the expectation that the values for the amino acid residues might prove to be somewhat low. The most highly purified bacitracin A gave a hydrolysate with practically no color under these conditions. The excess of hydrochloric acid was removed by evaporation under reduced pressure and the residue was dissolved in water. This was the standard solution used for quantitative analysis on the ion exchange column. When referred to the weight of peptide t.aken and after correction for the solvent loss on drying at 100” at 0.2 mm. pressure, the total nitrogen (Kjeldahl) was found to be 15.8 per cent. The Van Slyke amino nitrogen was found to be 13.6 per cent and the ninhydrin carboxyl nitrogen 12.76 per cent. The amino acids were determined quantitatively by the ion exchange