Investigations on ficin : III. Purification of ficin by gel filtration and the characterization of other protein fractions of ficuslatex
✍ Scribed by Giovanni Porcelli
- Publisher
- Elsevier Science
- Year
- 1967
- Tongue
- English
- Weight
- 364 KB
- Volume
- 28
- Category
- Article
- ISSN
- 1873-3778
No coin nor oath required. For personal study only.
✦ Synopsis
After earlier success in the column chromatographic purification of ficin on CM cellulose and then DEAE celluloser, attention has now been ,turned to a technique permitting not only the purification of ficin, but also the characterization of other protein fractions present in Ficus latex, used as a proteolytic agent in certain coun-tries2. The choice fell on gel filtration, an efficient technique for the separation of biologically active proteins".
EXPERIMENTAL AND RESULTS
Ten grams of a powder obtained in the crude form from the latex of Ficws amWzeZmirttica* were dissolved in zoo ml of water, 60 g of ammonium sulphate were added, and the solution was allowed to stand for L h at 5". The resulting precipitate was separated off by centrifuging the solution at xo,ooo r.p.m. for LO min, washed three times with a 23% solution of ammonium sulphate, redissolved in ~$0 ml of water, and dialysed in a cold-room for 48 h with zo 1 of distilled water. The resulting precipitate was removed by successive centrifuging, and, on being rendered lyophilic, the remaining clear solution gave about 5 g of pure sa.mple. 50 mg of, the sample were dissolved in 5 ml of a 0.005 M tris-phosphate buffer '(pH 7.2,0.g y0 of NaCl), placed on a go x 3 cm column packed with Sephadex G-100, and eluted with the same buffer at an average flow rate of 30 ml/h. The eluent fractions were monitored spectrophotometrically for proteins, which showed an extinction maximum at 280 my (broken curve in Fig. IA). A 400 ~1 portion of each fraction was then incubated at 37" for I h with z ml of a z y0 casein solution, the .proteins were precipitated with 5 y0 trichloroacetic acid, centrifuged, and the supernatant liquid was used to determine the extinction at 280 rnp. The enzymatic activity of the various eluent fractions, found in this manner, is shown by the full curve in Fig. IA.
Whereas the broken curve (protein concentration) exhibits five peaks (and an inflexion high up in the central peak), the fuli curve (enzymatic activity) stops after the large central peak coinciding with the third and highest maximum in the dashed curve, and, therefore, this is attributed to ficin.