A micromethod involving the use of fluorescent derivatives of 1-dimethylaminonaphthalene-5-sulphonyl chloride (Gray & Hartley, 1963) has been used in a parallel study of the structure of peptide C8Pla (as defined by Ambler, 1963) of the Pseudomonas cytochrome c-551. The basis of the method was a modification of the phenyl isothiocyanate procedure for the removal of the N-terminal residue (Edman, 1950 a, b), followed by investigation of the free amino groups of the residual peptide The very high sensitivity of the fluorescent end-group reagent used enabled the subtractive procedure to be applied over six consecutive steps, commencing with approx. 0-02 timole of peptide. The partial sequence Gly(Pro2,Ileu)-Met-Pro-Pro-Asp(NH2)-Ala had already been determined by cyanogen bromide cleavage and partial acid hydrolysis (Ambler, 1963).The peptide (0-02 ,imole) was oxidized with performic acid, to convert methionine into its sulphone. The oxidized peptide is referred to below as CPO, and in subsequent stages of degradation as CPOb1, CPO,2, according to the convention adopted by Ambler (1963). Interaction of peptides with the sulphonyl chloride attaches the fluorescent dimethylaminonaphthalenesulphonyl residue to free amino groups.Abbreviations. The 1-dimethylaminonaphthalene-5-sulphonyl derivatives are indicated by the term DNS-.Phenyl isothiocyanate degradation. The oxidized peptide (CPO) was dissolved in 0 1 ml. of water in a 5 ml. stoppered tube, and 10 ul. was removed for end-group studies. To the remainder was added 0-2 ml. of a solution of phenyl isothiocyanate in pyridine (5 %, v/v). The resulting single-phase mixture was incubated overnight at room temperature, and, after the addition of 0-2 ml. of water, the pyridine and excess of phenyl isothiocyanate were removed by four extractions with 3 ml. of benzene.The aqueous solution was then freeze-dried in situ, and 0-2 ml. of acetic acid saturated with anhydrous hydrogen chloride was added. After 30 min. at room temperature, the acid was removed in vacuo and the residual peptide dissolved in 0-1 ml. of water. A portion corresponding to one-tenth of the original material was removed for end-group studies, and the remainder resubmitted to the degradation procedure. At each successive cycle a similar portion was removed.N-Terminal studies. Samples from each cycle of the phenyl isothiocyanate degradation, corresponding to approx. 2 ,um-moles, were placed in small fermentation tubes containing 10 ,u. of 0 1 Msodium hydrogen carbonate in ammonia-free water.To each of these was added 15 ,l. of a 1 % (w/v) solution of 1-dimethylaminonaphthalene-5-sulphonyl chloride in acetone, forming a single-phase reaction mixture. After 2 hr. at room temperature, during which time all excess of reagent had been hydrolysed to sulphonic acid, the mixture was applied as a 2 cm. band to a strip of WVhatman no. 3 MM paper. High-voltage ionophoresis was then carried out for 1-5 hr. at 120 v/cm., with the apparatus describedby Gross (1961). The buffer used was 10 % (v/v) ofpyridine in 0 4 % ace...

Bovine pancreatic juice contains approximately equal amounts of four inactive precursors of endopeptidases (zymogens): chymotrypsinogen A, chymotrypsinogen B, trypsinogen (Keller, Cohen & Neurath 1958) and a component of procarboxypeptidase which resembles a chymotrypsinogen (Brown, Greenshields, Yamasaki & Neurath 1963). In porcine pancreas another endopeptidase, elastase, is found which is uniquely effective against elastin, the elastic protein of ligaments. Chymotrypsin A and chymotrypsin B are almost identical in enzyme activity (Enenkel & Smillie 1963), but the chymotrypsins, trypsin and elastase have widely different substrate specificities, as seen, for example, in their action on the B chain of oxidized insulin (Naughton & Sanger 1961; figure 1).