Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography
✍ Scribed by Chi-Sun Wang
- Publisher
- Elsevier Science
- Year
- 1980
- Tongue
- English
- Weight
- 611 KB
- Volume
- 105
- Category
- Article
- ISSN
- 0003-2697
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✦ Synopsis
Sequential chromatography of human milk whey on concanavalin A-Sepharose 4B followed by cholate-Sepharose 4B yielded a bile salt-activated lipase with M-fold purification. The lipase was not retained by concanavalin A-Sepharose 4B but was retained by the cholate-Sepharose 4B, from which it was eluted with 2% sodium cholate. The aflinity chromatography procedure on cholate-Sepharose 4B was based on the specific structural requirement of the enzyme for a 'I-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on ureasodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 05 1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.