Amino Acid Sequences of Peptides from the Tryptic Digest of Golfingia gouldii Hemerythrin^*

Abstract

Peptides from two separate tryptic digests of Golfingia gouldii hemerythrin were purified by Dowex 50W-X2 column chromatography followed by paper electrophoresis, paper chromatography, gel filtration, or various combinations of these techniques. The amino acid sequences of these peptides were completely determined, except for the relative positions of some of the residues in three peptides. These sequences account for a total of 113 residues T Xhe preceding paper (Groskopf et al., 1966) gives the amino acid composition as well as the aminoterminal and the carboxyl-terminal sequences of Golfingia gouldii hemerythrin. The present paper will describe the amino acid sequences of all the peptides recovered from tryptic digests of the protein. Experimental SectionPreparation of Tryptic Digests. G. gouldii hemerythrin was prepared and crystallized, and the ironfree protein was recovered, as previously described (Groskopf et al., 1966). Two separate tryptic digests, one from the whole protein and the other from the iron-free protein, were utilized.In the first case, 2.5 g (179 /¿moles of subunit) of oxyhemerythrin, dissolved in 435 ml of pH 8.0, 0.02 m ammonium bicarbonate, was denatured by heating at 65°for 5 min. The resulting suspension was cooled, and to it was added 25 mg of trypsin (Worthington Biochemical Corp.) treated with dilute hydrochloric acid according to Northrop and Kunitz (1936) to destroy residual chymotryptic activity. Further additions of 25 mg of trypsin were made at 4 and 8 hr and the hydrolysis was allowed to proceed at 32°for 22 hr. The digest was lyophilized. The peptides recovered from this digest are denoted T.

The aminoterminal sequence of Dendrostomum pyroides hemerythrin

“…It therefore appears that residue 9, which is valine in the Golfingia protein, is occupied by glycine in the Dendrostomum hemerythrin, a change which is consistent with a single mutation. The present work indicates that residues 10 and 1 1 are tryptophan and aspartic acid respectively in both Golfingia and Dendrostomum hemerythrins and suggests that these residues were inverted in the earlier report of the Golfingia sequence [2,4].…”

“…The sequence determined for the aminoterminal region ofD. pyroides hemerythrin is given in table 1 together with the sequence determined by Klotz and coworkers [1][2][3][4] for this portion of the pigment from Golfingia gouldii. These sequences are identical except for residues 9, 10 and 11.…”

“…The pigment has a molecular weight of 107,000 and is composed of 8 subunits. The only sequence information available is for the subunit of the hemerythrin from the sipunculid worm Golfingia gouMii [1][2][3][4]. We have described earlier the properties of the hemerythrin from the sipunculid Dendrostomum pyroides [5 ] and now report the sequence of the first 35 aminoterminal residues of this protein.…”

Selective deamidation and enzymic methylation of seminal ribonuclease

“…The question of whether the isoaspartyl residue is at position 67 or 71 in these peptides could not be solved directly, for the lack of sequence data in the region of interest (see above). However, we can confidently locate the isoaspartyl residue of peptides «14 and «16 at their position 67, on the basis of the resistance of bond 66-67 of these peptides to trypsin and leucine aminopeptidase and of the resistance to cleavage by the Edman reaction of bond 67-68. It is well-known that (1) trypsin hydrolyzes very slowly peptide bonds adjacent to «-carboxyl groups (Neurath & Schwert, 1950; Waley & Watson, 1954), (2) leucine amino peptidase does not cleave isoaspartyl /3-peptide bonds (Delange & Smith, 1971), and (3) cyclization to thioazolinone and cleavage do not occur in the Edman reaction when the NH2-terminal residue is an isoaspartyl residue (Groskopf et al, 1966;Weber & Konisberg, 1967).…”

A Low Molecular Weight Proinflammatory Factor from Rat Spleen Lymphocytes. Isolation and Partial Characterization

“…Spots above and below DNS-Glu are DNS-NH 2 and DNS-OH, by products of the dansylation procedure. occurred, suggesting gGlu at the N-terminal position, since it is known that the peptide linkage involving the g carboxyl groups of Glu is resistant to splitting by phenyl isothiocyanate [29]. As the same occurred with GSH (gGluYCysYGly) and GSSG, our attention was once again directed to glutathione, knowing however that it displayed no proinflammatory activity.…”