Structure and Action of Heteronemertine Polypeptide Toxins DISULFIDE

The positions of the disulfide bonds in Cerebratulus lacteus toxin B-IV were investigated by hydrolysis of the unre- duced protein with a variety of proteases. The resulting peptides were purified by gel filtration, ion exchange chro-matography, and preparative paper electrophoresis and chromatography. Determination of the amino acid composi- tions of the cystine-containing peptides purified demon-strated the existence of disulfide bonds linking half-cystine residues 12 and 24,21 and 51, and 35 and 38. The fourth bond, involving half-cystines 10 and 47, was assigned by differ-ence. acid

The amino acid sequence of toxin B-IV, a crustacean-selective, axonal neurotoxin purified from the heteronemertine worm Cerebrcztulus lacteus, has recently been established (1). The purpose of the experiments reported herein was to determine the positions of the disulfide bonds of toxin B-IV and to possibly gain some preliminary insight into the role of these bridges in the biological activity of the protein. Experimental procedures and details of the methods employed for purification of individual peptides are given in the miniprint section. ' RESULTS Toxin B-IV, the amino acid sequence of which is given in Fig. 1 ture of trypsin, chymotrypsin, and thermolysin. The positions for two of the four disulfide bridges could be determined by amino acid analysis of the resulting peptides, the purification of which is detailed in the miniprint section. To obtain the positions of the remaining bridges, a second sample of the native toxin was maleylated and treated as described in the miniprint section. Peptides containing three of the four toxin disulfide bonds were purified and characterized in at least one of the hydrolysates described.
Identification of Disulfide Pairs-The sequences of the isolated cystine-containing peptides are shown in Fig. 2. In all cases, the residues of half-cystine contributing to a disultide pair could be identified from the amino acid composition of the isolated peptide (Tables I and II). However, in the case of Peptides (T-l,C-l,)-ThLC and -ThSB (Table II) a single cycle of the Edman degradation was run to confirm that alanine was indeed NH,-terminal.
Analysis of the <PTCY derivatives liberated, after hydrolysis to the free amino acid (lo), yielded only alanine. A second aliquot was analyzed by thin layer chromatography, after conversion of the anilinothiazolinone to the <PTC derivative. These samples contained Ala<PTC and an unidentified substance which failed to move off the origin in either heptane:l-butanol:formic acid (50:30:7, by volume (8)) or in xylene:2-propanol (7:2, by volume (9)).

DISCUSSION
One of the more difficult problems encountered in the course of this work was the extreme resistance of native toxin B-IV to the commercially available proteases. Pretreatment of the toxin with 8 M urea, 6 M guanidine HCl, by boiling, or by acidification to pH 2.3, failed to relieve this intractibility. Hence, after an inital reluctance to expose the native toxin to neutral or slightly alkaline pH, succinylation and maleylation were employed; either procedure served to render the protein susceptible to trypsin. No evidence for disulfide exchange was observed during this study in that no cystinyl peptides having compositions incompatible with our disulfide assignments were purified.
The procedure used for maleylation of the protein is worthy of comment.
Our intent was not quantitative modification of p The abbreviation used is: <PTC, phenylthiohydantoin.   Tables I and II. In each case, a single peptide is shown to prove the existence of the bridge, although variants of both SMP-3D and T-l,C-1, Th2C were also characterized. the amino groups; rather, we hoped to modify a sufficient fraction of these groups to render the protein sensitive to brief treatment with trypsin. This limited hydrolysis should then serve to denature the protein, leaving all of the lysine and arginine peptide bonds sensitive to trypsin, after demaleylation. For these reasons, a smaller than normal excess of maleic anhydride was utilized, the reaction was carried out for 5 min at pH 8.0, rather than 9 to 10, and tryptic hydrolysis ensued immediately, without prior removal of free maleic acid. Additionally, exposure of the maleylated protein to trypsin was brief (40 min) and carried out at pH 7.0. Our hopes were realized, in that the bulk of trypsin-sensitive bonds were apparently cleaved, and no evidence for disulfide exchange was observed.
The application of trypsin, chymotrypsin, thermolysin, and elastase, either singly or in groups, allowed isolation and characterization of peptides containing three of the four disulfide bonds known to exist in toxin B-IV. Thus, the evidence for the bond linking Cys-10 and Cys-47 is indirect. In view of the known participation of all 8 residues of half-cystine in the protein in disuliide bonds (2), the lack of evidence for disulfide exchange, and since the remaining three disultide pairs have been firmly established herein, the existence of a Cys lo-Cys 47 disulfide is at least highly probable. While it would have  (1) 1.7 (2) 2.2 (2) 0.9 (1) 0.4 (0) 2.0 (2) 0.8 (1) 1.3 (1) 1   (1) 1.0 (1) 10 0.68 been more desirable to have direct confirmation of this bond, we felt that the large amount of toxin which might be required for confirmation could be more usefully employed for chemical modification studies. This paper describes the first investigation of the disultide bonds of heteronemertine toxins. It may therefore be worth noting that the disuhide bond pattern established is different from that observed with scorpion (11) or snake neurotoxins (12). As noted elsewhere (1) primary structures of scorpion, snake, and Cerebratulus toxins display no homology. One further interesting point is raised by our findings. The disulfide bond linking Cys-35 and Cys-38 must result in the formation of a hairpin turn in the polypeptide chain (residues 35 to 38) which can be predicted by the method of Chou and Fasman (13).3 It would be of interest to ascertain whether the Cys 35-Cys 38 pair can be preferentially reduced and to determine the effects of such partial reduction upon toxicity, particularly since current theory implicates a carboxylate group in the function of the sodium channel (14, 151, and the loop formed as a consequence of the hairpin should be highly cationic (Fig. 1). Experiments studying the effects of partial reductions are currently in progress.