. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. BRAIN HORMONE PURIFICATION 363 ^ o CO fM m tr o en. A 3x10' i— 2x10' = x 1x10' • 20 60 TUBE NUMBER 60 FIGURE 5. Gradient chroinatography on DEAE-cellulose. to further purification. This fraction is designated as DEAE-Cellulose 6-16 Fraction. One may notice that the total activity of BH illustrated in Figure 5 (248,000 units) plus the activity in the washing (80,000 units) is much smaller than that of the starting material in this chromatography (1,280,000 units). However, the first assay employed here implies a possib
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. BRAIN HORMONE PURIFICATION 363 ^ o CO fM m tr o en. A 3x10' i— 2x10' = x 1x10' • 20 60 TUBE NUMBER 60 FIGURE 5. Gradient chroinatography on DEAE-cellulose. to further purification. This fraction is designated as DEAE-Cellulose 6-16 Fraction. One may notice that the total activity of BH illustrated in Figure 5 (248,000 units) plus the activity in the washing (80,000 units) is much smaller than that of the starting material in this chromatography (1,280,000 units). However, the first assay employed here implies a possibility that each of the negative fractions might have contained up to 4000 units BH. If all of the 59 negative fractions had contained 4000 units, then 4000 X 59 = 236,000 units could have been present in these fractions. A sum of all of the above is 564,000. In view of the experi- mental error necessarily involved in the assay method using 2-fold-dilutions, the difference between 1,280,000 and 564,000 units is in the range of the experimental error. But the above calculation is based upon the assumption that the maximum possible amounts were present in all of the negative fractions. The actual amount in the negative fractions is possibly much smaller; in that case the total amount of BH recovered is too small to ascribe to the experimental error. It is possible that partial inactivation of BH occurred in this purification step. Second gel-filtration on Sephadex G-100 column. The DEAE-Cellulose 6-16 Fraction was precipitated with ammonium sulfate, with pH adjustment at — with N NaOH. The precipitate was dissolved in 3 ml. of M Tris-HCl, pH , and dialyzed against the same buffer. This solution was again subjected to gel-filtration on a X cm. column of Sephadex G-100. The elution was performed with the above buffer at a flow rate of 30 Fractions were collected each having a volume of 9 ml. Bioassay was again carried out in two steps. In the
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
