THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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Fractioñâtión of oPL. from crúde extrác-t by ácidificgtign
Although fractìonal precìpitation by lowering the pH of the
crude extract was not the most effectlve method( M.Sc. Thesfs,l975),
lowering the pH of the crude extract to 6.5 with.,qlacial acetic acid resulted in
precipitation of .. some mucous substances whjch Jnterfere with the
subsequent anion-exchange chromatography. Uslng thÍs procedure, I couìd
eliminate about I5% of the original protein content, but reta.ined about
95% of the original gi^owth hormone activity in the extract.
Di ethyani noeth.yl ( DEAE ) -cel I u I ose an i on exchangr__g¡-Ig¡g!9gl9-p¡y.
Beforè applyingthe acidified extract to DEAE-ion exchange
chromatography, the extract was re-adjusted to a pH of 9.0 with
2N anrnonium hydroxlde. At this pH, nost of the opl was unabsorbed by the
column, whereas a considerable amount of protein was absorbed. l{ith
lncreasing concentrat.iors of NaCì.in the presence of 0.01 M glycine-OH,
pH 9.0, Iittle addìtionaì hormone was eluted as shown in Figure 5. In
practice one need not elute with higher salt concentratìon.
Carboxylnrethvl (CM)-cel'luìose cation exchanqe chromatography
The fractions containing opl after DEAE coìumn chi"omatography were
acidified to pH 6.0, centrifuged to remove the prec.ipìtates,
and appl ied to a CM-cellulose column for additÍonal purification ag
shown in Figure 6. ülhen a stepwise sart gradient ( 0.05M to 0.5M NacL)
was enpìoyed to elute the opl fron the coìumn, most of the opl was e'ruted
by 0.2M NaCL. Only a smaì.I anount of opl was eluted
with 0.5 M NaCL. The O.2M fractions were then further fractionatedon the
Cqrboxymethyl -Sephadex column .