Active fragments obtained by cyanogen bromide cleavage of ovomucoid

Abstract

Cleavage of the two methionine residues in the glycoprotein trypsin inhibitor ovomucoid, variant 01, with CNBr resulted in two fragments whose mol.wts. were approx. 16 600 (fragment LS) and 11000 (fragment M). Both fragments formed precipitates with antisera to ovomucoid. Fragment LS retained 56 % of the trypsin-inhibitory activity of ovomucoid, but fragment M did not inhibit. After reduction and alkylation, the molecular weight of fragment M was unchanged, but fragment LS could be resolved into two segments of peptide chain with mol

Location of the carbohydrate groups of ovomucoid

“…Amino acid analyses were performed after hydrolysis at 1 10°C for 24h in 6M-HCI in evacuated sealed tubes containing lOO1g of phenol/ml. Hexosamine was determined by using an amino acid analyser after hydrolysis with 4M-HCI for 3h at 100°C or with 3M-methanesulphonic acid at 100°C for 24h (Beeley, 1976). Amino acid and hexosamine determinations were related by the inclusion of norleucine as internal standard in both determinations.…”

“…Eggs were obtained from individual hens and sialic acid-free ovomucoid variant 01 was isolated as described previously (Beeley, 1976). All preparations used gave a single band on gel isolectric focusing (Beeley, 1971), indicating that they were homogeneous in charge.…”

“…Subtractive Edman degradation yielded the sequence of nine residues of peptide T-4a ( Fig. 4) which had thie composition shown in Table 1 (Beeley, 1976). in loss of aspartic acid, and analysis of the unhydrolysed products indicated that threonine was the only detectable free amino acid.…”

“…The isolation of three reduced aminoethylated CNBr fragments of ovomucoid (Li-AE, M-AE and S-AE) and the evidence establishing their order of occurrence in the peptide sequence of ovomucoid have been described previously (Beeley, 1976).…”

“…To do this, tryptic pep-Vol. 159 tides obtained from reduced, alkylated ovomucoid or CNBr fragments of ovomucoid (Beeley, 1976) were fractionated and sequenced. The ovomucoid used was the major variant ovomucoid 01 (Beeley, 1971), which is free of sialic acid and was obtained from the eggs of an individual hen.…”