Elution patterns DEAE-Sephadex

Fractionation and Purification of Ex-1 Cellulase Component from Driselase. Driselase powder (50g) was extracted with several aliquots of water and the precipitate formed upon salting out with ammonium sulfate (on a saturation between 20% and 80%) was fractionated on a DEAE-Sephadex A-50 column. Each fraction was tested for -glucosi-dase, xylanase, CMCase, Avicelase activities, and protein content. The elution patterns are shown in Figures 1 and 2. 

Figure 1. Elution patterns of the ammonium sulfate-precipitated enzyme preparation from a DEAE-Sephadex A-50 column. (0) CMC-saccharifying activity (5-min incubation) of eluates diluted 60-fold, (O) Avicel-saccharifying activity (1-hr incubation), ( ) protein concentration measured in terms of the absorbance at 280 nm column 5.0 X 50 cm flow rate 20 mL/8 min one fraction 20 mL.

Amir et al. employed DEAE-Sephadex A-50 in a final purification step for human FSH however, they lost 60% of the biological activity when they subjected their material to this procedure. They stated that the elution pattern of the proteins from DEAE-Sephadex was not reproducible because the LH, FSH, and albumin tended to form complexes which varied in their behavior on the column. This explanation should be accepted with reserve (A7). 

Proteinase A was purified from yeast extracts, activated by incubation at pH 5, by repeated chromatography on DEAE-Sephadex followed by alcohol precipitation, as described by Hata et al. (3). The enzyme could be further purified in our laboratory by affinity chromatography of the partially purified enzyme, using the specific proteinase A-inhibitor I, bound to activated CH-Sepharose 4B as the stationary phase. The pattern of elution by 6 M urea from the affinity column is shown in Figure 1 the peaks of hemoglobinhydrolyzing activity (i.e. proteinase A activity) and protein concentration coincide. Table II summarizes the present purification procedure for proteinase A. Work on further purification and characterization of the enzyme is in progress. 

Figure 19. A Gel filtration pattern of a 40X1 tryptic hydrolysate (1 hr) of bovine serum albumin. The hydrolysate (0.3 g) was applied onto a column (2.7 x 80 cm) of Sephadex G-100 which was eluted with 0.01 M NH HCOs. Fractions (7.5 ml each) were analyzed continuously by an ultraviolet monkor. B Chromatogi hic pattern on DEAE-cellulose of peak 3 from A. The material (100 mg) was applied on a column (1.5 x 20 cm) which was subjected to stepwise elution. Initial elution was with 0.005 M sodium phosphate buffer at pH 6.2. At positioa 2 the column was eluted with 0.0175 M phosphate buffer pH 6.2, and at portion 3 the ehient was changed to 0.0175 M phosphate pH 6.2 plus 0.077 M NaCl. Fractions (3 ml) were read continuously by an ultraviolet monitor. From Habeeb and Atassi (19766).

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