. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 362 HIRONORI ISHIZAKI AND MAMORI ICHIKAWA BH UNIT FRACTION POOLED- « 20,000 it any UNABSORBED •• 15,000 if any 0 KM 74,000 1,280,000 M « NoCI 50 100 150 TUBE NUMBER FIGURE 4. Stepwise chromatography on DEAE-cellulose. Tris-HCl buffers (pH ) of the specified molarities were applied at the places designated by arrows. Brackets represent the pooled fractions. were collected. The effluents at each step were pooled and BH units contained in them were determined. The results are shown in Figure 4. The
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 362 HIRONORI ISHIZAKI AND MAMORI ICHIKAWA BH UNIT FRACTION POOLED- « 20,000 it any UNABSORBED •• 15,000 if any 0 KM 74,000 1,280,000 M « NoCI 50 100 150 TUBE NUMBER FIGURE 4. Stepwise chromatography on DEAE-cellulose. Tris-HCl buffers (pH ) of the specified molarities were applied at the places designated by arrows. Brackets represent the pooled fractions. were collected. The effluents at each step were pooled and BH units contained in them were determined. The results are shown in Figure 4. The most active fraction was the M fraction which contained 1,280,000 units BH. The M fraction and M + M NaCl fraction contained 74,000 and 64,000 units, respectively. The unabsorbed fraction and M fraction turned out negative when tested by injecting ml. into each assay pupa, indicating that they contain less than 20,000 and 15,000 units, respectively. The M fraction was subjected to subsequent purification and is designated as DEAE-Cclhdose M Fraction. Gradient chromatography on DEAE-cellulose. The DEAE-cellulose M Fraction was dialyzed against M Tris-HCl, pH , and placed on a X cm. column of DEAE-cellulose equilibrated with the above buffer. The column was washed with the buffer and gradient chromatography carried out between 300 ml. each of M and M Tris-HCl, pH The flow rate was 35 and 8-ml. fractions were collected. Because of the limited number of assay pupae available, the bioassay of 75 fractions was carried out in two steps as follows. First, aliquots from all fractions were diluted 20 times with deionized water, and assayed by injecting ml. into each of the assay pupae. By this assay one can distinguish fractions which contain more than 8,000 units BH by their positive responses. After knowing the result of the first assay, the positive fractions only were again assayed to determine the
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
