The sequences of the tryptic peptides from actinidin : a thesis presented in partial fulfillment for the degree of Doctor of Philosophy [in Biochemistry] at Massey University

Abstract

Actinidin is a plant thiol protease which is isolated from the fruit of Actinidia chinensis, the chinese gooseberry. Determination of the primary amino acid sequence of actinidin was undertaken to extend the limited structural information available on this group of enzymes, and therefore enable a better understanding of their physical and chemical properties. The order of arrangement of the 220 amino acid residues in the primary sequence of actinidin was determined from the sequences of the tryptic peptides. S-carboxy[14C2]methyl actinidin was digested with trypsin, and the twelve tryptic peptides produced were initially separated into seven fractions by gel chromatography on Sephadex G-50. The first four fractions contained tryptic peptides that were purified by DEAE-cellulose chromatography. The last three fractions contained peptides that were sufficiently small to enable purification by paper techniques, and these peptides were sequenced directly by the dansyl-Edman method. Further degradation of the tryptic peptides purified on DEAE-cellulose with either chymotrypsin, thermolysin, pepsin or Staphylococcus aureus V8 protease was necessary to provide smaller peptides that could be sequenced by the dansyl-Edman method. S. aureus V8 protease was particularly useful in the determination of amide residues, because of the enzyme specificity for the carboxyl groups of glutamic acid. The fourth tryptic peptide in the sequence of actinidin could not be located in the tryptic peptide elution profiles of either the Sephadex G-50 or DEAE-cellulose columns. The sequence of this peptide was determined from a tryptic peptide obtained by digestion of maleylated carboxymethyl actinidin. The N-terminal of actinidin was determined by the dansyl Edman method, and the C-terminal by analysis of cyanogen bromide fragments, and by digestion with carboxypeptidase A. Radioactively labelling the active site cysteine residue with iodo[14C2]acetic acid, and subsequent purification of the radioactive tryptic digest peptide, enabled the isolation of the tryptic peptide containing the active site cysteine residue. Further digestion of this peptide with chymotrypsin and determination of the sequence of the smaller radioactive peptide provided the sequence about the active site cysteine residue. Alignment of the tryptic peptides to reconstruct the primary sequence of actinidin was accomplished with information from cyanogen bromide fragments, information from tryptic peptides of maleylated carboxymethyl actinidin, and information from the three dimensional X-ray crystallographic structure of actinidin determined by Dr E.N. Baker. The low proportion of basic residues and high proportion of acidic residues in actinidin are in agreement with the enzyme being an acidic protein. Colorimetric analysis of the tryptophan residue content, using 2-nitrophenylsulphenyl chloride, confirmed the presence of six tryptophan residues in the sequence of actinidin. The amino acid sequences about the seven cysteine residues and the single histidine residue in actinidin were very similar to the analogous sequences in papain and other plant thiol proteases. Furthermore, comparison of the primary sequence of actinidin with that of papain, and the fragments of sequence available for other plant thiol proteases, indicated a considerable homology throughout the sequences of these proteins.