2% - n V . a; . ’ :Ewnéi. l . :z 9 .— I: -I III J , I 1_oo T l l l l l II I I l l I I II T .104 105 Il°° MOLECULAR WEIGHT Figure 5. The standard curve used for assigning molecular weights of proteins as determined by applying the proteins to a 2 x 80 cm column of G-ZOO Sephadex and measuring the volume required to elute the proteins. Extracellular toxin eluted as indicated by the arrow. 29 r. l I l— l i__1_.0 Ls L..6 A280nm LA I l 1 L2 1 T E I l I I 20 40 60 80 160 ML ELUTION Figure 6. Absorbance at 280 nm of elutant from a 1 x 28 cm column of DEAE cellulose after application of four m1 of concen- trated type E C. botulinum toxin followed by elution with 0.05 M sodium phosphate buffer at pH 6.0 (the toxin eluted with the major absorption peak). 30 most of the protein and toxin. This peak was followed by a small snoulder or trailing peak which contained little toxin. The toxic fractions were pooled, concentrated using ultra- filtration with an Amicon UM-2 membrane and applied to a C-50 Sephadex column. A large frontal peak eluted first, followed by low absorption (Figure 7). The large peak usually included a small amount of toxin, but most of the toxin adsorbed to the column. The adsorbed toxin could be eluted from the column by the addition of 0.5 M NaCl to the phOSphate buffer. Use of a linear salt gradient from 0.0-0.5 M NaCl produced a spreading of the absorbant peak con- taining the toxin resulting in a leading shoulder on the main absorbant peak (Figure 8). Three separate fractions from a spreading absorbant peak from a C-50 Sephadex column, such as that shown in Figure 8, were assayed for toxicity, analyzed for protein content and applied to a pH A.3 polyacrylamide gel electrophoresis system. These fractions were found to have different toxicities and different protein contents. Figure 9 illustrates the fractions of the absorbant peak and the electro- pnoretic patterns obtained from the three fractions which were assayed. Fraction A7 contained 3 distinct bands. The band with the lowest mobility was labelled B], the next lowest, 82, and the band with the highest mobility, B3. Fraction A5 contained 2 significant bands, equivalent to BI and B3, and Fraction 50 revealed only one band, 83. The protein concentrations and specific toxicities of these fractions are shown in Table 3. Since fraction A5 had a higher specific toxicity than either fraction A7 or 50, band 83 which was the most intense band in both 31 A230...». Figure 7. I I I I 10 20 30 40 ML ELUTION A sample of extracellular toxin, after elution through a column of DEAE cellulose, was applied to a l x 28 cm C-SO Sephadex column and eluted with 0.05 M sodium phosphate and monitored at 280 nm (a low level of toxin was present in the absorbance peak). 32 .25 __4 AQBOnm __2 _.J l I I’ f ‘ I 18 20 3% 40 ML OF NuCI GRADIENT Figure 8. Adsorbed toxin, eluted through a column of DEAE cellulose and adsorbed onto a l x 28 cm column of C-50 Sephadex was eluted with a 0-0.5 M NaCl gradient in 0.05 M sodium phosphate buffer at pH 6.0. (The toxin eluted before the 280 nm absorbant peak.) Figure 9. The fractions collected during the elution of the extracellular toxin adsorbed to a C-50 Sephadex column with a 0-0.5 M NaCl linear gradient in 0.05 M sodium phosphate buffer at pH 6.0, and the protein bands from three of the fractions after electrophoresis in 72 polyacrylamide gel at pH A.3 and stained with Buffalo Black stain. 3A Table 3. Specific toxicity and protein concentration of three fractions from the adsorbed extracellular toxin eluted from a C-50 Sephadex column. Fraction No. Hg protein/ml MLD/mg protein* A5 67 3.0 x 106 117 618 1.6 x105 so 223