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2 R. Ros s , in B. S. GOULD, Treatise on Collagen, Vol. 2A, Academic Press, New York, 1968, p. i . 3 J. A. CHAPMAN, in G. E. W. WOLSTENHOLME AND M.O'CoNNOR, Principles of Biomolecular

Organisation, Churchil l , London , 1966, p. 129, 4 D. S. JACKSON AND IL. G. CLEARY, in D. GLICK, Methods of Biochemical Analysis, Vol. 15,

Interscience, New York, 1967, p. 25. 5 G. C. WOOD, in D. A. HALL, International Reviews of Connective Tissue Research, Vol. 2,

Academic Press, New York, 1964, p. I. 6 G. C. WOOD, Biochem. J., 75 (196o) 598. 7 J. C. CAYGILL, Biochim. Biophys. Acta, 17o (I968) I. 8 B. P. TOOLE AND D. A. LOWTHER, Biochem. J., lO 9 (1968) 857. 9 J. GROSS, Nature, 181 (1958) 556.

io J. J. REYNOLDS, Exptl. Cell Res., 47 (1967) 42. 11 Y. SHIMIZU, D. S. McCANN AND M. K. KEECH, J. Lab. Clin. Med., 66 (1965) 659. 12 B. S. GOULD, in B. S. GOULD, Treatise on Collagen, Vol. 2A, Academic Press, London , 1968,

p. 323 • 13 R. A. WELSH AND A. T. MEYER, Arch. Pathol., 84 (1967) 354. 14 D. J. PROCKOP AND t~. I. KIVIRIKKO, in ]3. S. GOULD, Treatise on Collagen, Vol. 2A, Academic

Press, London , 1968, p. 215.

Received December 3rd, 1968

Biochim. Biophys. Acta, 181 (1969) 334-336

BBA 33148

Dissociation studies on the toxin of Clostridiurn botulinum type B

I t has been shown that a major toxic product present both intracellulafly z and in lysates ~ ofClostridium botulinum type B is a macromolecular structure with s == 15. We have reported the presence of lysates of low molecular weight toxic moieties s. I t has recently been shown that this I5-S toxic component can be dissociated under alkaline conditions and the dissociation products isolated by chromatography on DEAE-cellulose 4, one product possessing approx. 5 times the specific activity of the original product, and designated by the authors as the Ba component. This report constitutes a study on the chemical nature of these dissociation products in an a t tempt to determine whether two distinct protein species are present in the I5-S material and whether or not a chemical relationship exists between these materials and low molecular weight toxic moieties reported earlier.

The methods for isolation of the I5-S toxin have been described previously 1. The pelleted material was dissolved in o.o 5 M acetate buffer (pH 4.5) at 5 mg/ml and yielded a single sharp peak of s = 15.5 in the analytical ultracentrifuge. End group analysis, by the method of GRAY AND HARTLEY 5 was carried out on this material and only arginine was detected. This is in agreement with end group analysis carried out previously on low molecular weight toxic materials 3. Since only one amino acid was detected no such tests were run on the dissociation products. The I5-S toxin was chromatographed under alkaline conditions by methods previously described 4, to yield the two major dissociation products, the Ba or highly toxic material, and the other poorly toxic material (designated here Peak I I DEAE). Amino acid analyses were carried out on a Beckman model 12o analyser 6 on the I5-S material, Ba and Peak I I DEAE. Three separate batches of material were prepared, processed and

Biochim. Biophys. Aeta, 181 (1969) 336-338

SHORT COMMUNICATIONS

T A B L E I

AMINO ACID COMPOSITIONS OF VARIOUS DISSOCIATION P R O D U C T S OF I 5 . 5 - S T O X I N Figures r ep resen t m e a n 4- S.E.

A mino acid Product (g amino acid/zoo g protein)

z5.5-S toxin Ba fraction Peak I I D E A E D E A E + G-2oo

Lys 6.90 4- 0.38 7.25 ± 0.4 6.50 ± o,42 His 0.72 + o.21 0.57 4- o.12 o.61 4" o,o4 Arg 2.85 4, o.12 2.27 4. 0.26 2.35 4. 0,27 Asp 17.67 4- 0.75 18.32 4. 1.45 19.62 4. o,73 T h r 4.46 4. 0.52 4.36 4- o.21 5.96 ± o,3I Ser 9.35 ± o.49 9.23 4. o.25 9.48 ± o,41 Glu 9.39 ± o.31 lO.22 ± o.57 9-55 ± o,22 Pro 3.78 4- 0.25 2.55 4- o.21 2.55 ± o.2o Gly 4.81 4" o.21 5.o3 -4- o.3o 4.7 ° 4" o,o7 Ala 4 .26 4" 0.27 4.32 4" 0.32 4.29 4" o,16 Cyg o.64 4" o.17 o.61 4" 0.28 o.51 4- o,o9 Val 4.78 4, o.21 4.24 4, 0.28 4.85 + o,23 Met 1.23 4- o.22 1.28 ± 0.22 1.o4 4" o,o9 Ile lO-31 -4- 0.52 9.94 -4:_ 0.53 9.37 4" 0,25 Leu 9.03 4- 0.35 8.30 4. o.36 8.35 4, o,21 T y r 6.26 4- o.31 5.35 + o.18 5.82 4- 0.50 Phe 4.95 4- 6.12 5.24 4- o. 15 4 . 4 2 4 " O,I5

337

hydrolyzed for this purpose, and each hydrolysate was analyzed in duplicate. Thus each set of data in Table I represents the average of 6 analyses. The data show that no definitely significant differences exist between the three preparations and that these analyses are in agreement with those carried out previously on low molecular Weight toxic materials 3.

Tryptic finger print studies were subsequently carried out in an at tempt to demonstrate possible differences between the dissociation products. Samples of I5-S toxin, Ba and Peak II DEAE at concentrations of 5 mg/ml were incubated in 8 M

% ~ ~ ~ ~ ~ ~ ~ s ~ / : ~ ~ ~ n H ~

Fig. I. T ryp t i c finger p r in t of I 5 , 5 - S toxin . Y indica tes a yellow area wi th n inhydr in .

Fig. 2. T ryp t i c finger p r in t of the Ba fract ion. Y indica tes a yellow area wi th n inhydr in .

Fig, 3. T ryp t i c finger p r in t of Peak II D E A E mater ia l . Y indica tes a yellow area wi th n inhydr in ,

Biochim. Biophys..4cta, 181 (1969) 336-338

338 SHORT COMMUNICATIONS

urea for 4 h at 37 ° and subsequently dialyzed for 18 h at 4 ° against distilled water. Trypsin (Worthington, 125 units/mg) was added to 2% (w/w), the pH was raised to 8. 5 with NHaOH , and digestion carried out for 2 h at 37 °. Digests were lyophilized and dissolved in formate-acetate buffer (pH 1.9) (2.5% formic acid-7.8 % acetic ac id v/v). Electrophoresis was run in this buffer for 30 rain at 3000 V. The second dimension was run chromatographically in pyridine-butanol-acetic acid-water (4 ° :60 : 12:48, by vol.) for 18 h. Finger prints were air-dried and stained with 1% ninhydrin in acetone and o.oi M cadmium acetate in 8 M acetic acid (85:15, v/v). The results are shown in Figs. I, 2 and 3. I t can be seen that close identity of peptide patterns exist between the three preparations under study. Also, the low number of peptides observed indicate the presence of a small subunit structure with at least large portions of the amino acid sequence common in all fractions. There does appear to be one (possibly two) extra peptide(s) in the Peak I I DEAE finger print when compared with Ba. Although these were not always reproducible, they do suggest some difference between the two fractions. These data suggest that I5-S material is made up of low molecular weight subunits. This is supported by the observation in this laboratory that I5-S material could be dissociated by dialysis against 0.2% sodium dodecyl sulfate with the formation of a product with s = 2.5 when studied in the ultra- centrifuge.

The difference in biological activity between Ba and Peak I I DEAE is definitely established 4, although the data indicate a common molecular species in both fractions. Thus one might explain differences in biological activity on the possibility of differing degrees of intermolecular bonding in the different fractions. Alternately, the Peak I I DEAE fraction may represent some kind of zymogen-like precursor since finger print analyses indicated the possibility of one or two more tryptic peptides in this fraction.

Department of Microbiology, The University of British Columbia, Vancouver (Canada)

BARBARA MITCHELL

JULIA GERWING

I J. GERWING, R. W. MORRELL AND R. M. NITZ, J. Bacteriol., 95 (1968) 22. 2 J. T. DUFF, J. KLERER, R. H. BIBLER, D. E. MOORE, C. GOTTFRIED AND G. G. WRIGHT, J .

Bacteriol., 73 (1957) 597. 3 J. GERWING, C. E. DOLMAN, D. V. KASON AND J. H. TREMAINE, J. Bacteriol., 91 (1966) 484 . 4 B. R. DAS GUPTA, D. A. BOROFF AND K. CHEONG, Biochem. Biophys. Res. Commun., 32 (1968)

lO57. 5 W. R. GRAY AND B. S. HARTLEY, Biochem. J., 89 (1963) 59. 6 D. H. SPACKMAN, W. H. STEIN AND S. MOORE, Anal. Chem.. 3 ° (1958) 119o.

Received December 9th, 1968

Biochim. Biophys. Acta, 181 (1969) 336-338