Dilute acid hydrolysis of papain (Part I), the amino acid sequence of dog heart cytochreom C (Part II)

Part 1: (i) Kinetic studies on the release of free amino from S-carboxy-methyl (SCM) papain by HC1 (0.03 to 0.003 M) at 110?, showed no increase in selectivity of release of free aspartic acid with decreasing HC1 concentration. (ii) A Grotthus-type mechanism is proposed to account for the lability of peptide bonds vicinal to aspartic acid residues. (iii) Peptides isolated from Dowex-50 column chromatographic fractions of hydrolysate of SCM papain of 0.03 M HC1 at 110? for 48 hr were characterized and their sequences investigated. (iv) The location of these peptides in the presently known extended sequences of papain established or confirmed several important overlaps in the sequence. (v) Extensive cleavage was found at peptide bonds vicinal to glutamic acid, glycine, alanine, threonine and serine residues as well as aspartic acid. The yields of the peptides investigated were as high as 20% but average below 10%. (vi) It is recommended that the use of dilute hydrolysis for obtaining peptides from proteins for sequence studies can be employed most usefully as an adjunct to enzymic degradation, to obtain bridge peptides, the existence of which has already been indicated and which can be located readily among the column chromatographic fractions of the hydrolysate by some relatively selective procedure. Part 2: (i) Dog heart cytochrome c has been isolated and characterized. It is homogeneous on electrophoresis and in the ultracentrifuge. (ii) the amino acid composition was determined. There are 8 differences in composition between dog and horse chtochromes c. (iii) From the overlapping compositions and sequences of 27 peptides each from chymotryptic and tryptic digests of dog heart cytochrome c, and by analogy with the sequences of the horse and human proteins, the complete amino acid sequence of the dog was deduced, and compared with those of the later two proteins. (iv) There are six amino acid replacements between dogs and horse cytochromes 47 (Ser), 60 (Gly), 88 (Thr), 89 (Gly), 92 (Als) and 103 (Lys). (v) The nature of these replacements and their significance for understanding both the relationship between structure and function in enzymes and the evolutionary relationship between species is discussed.