THE UNIVERSITY OF MANITOBA STUDIES ON OVINE PLACINTAL ...

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appropriate yo lu¡qe of Tris-HCl buff,er such that the protein
concentration in 0.1 mI, yo lume of the suspension contained
100 to 200 ug of protein as determined by the Lo!ì¡ry procedure
( Lowry et al | 1951) . I\,lembrane suspensions prepared f ro¡¡ one
rabbit Liver tissue provides sufficient receptors for 6,000
tubes .
2. Iodination procedure for hormone preparation (hunan growth
hormone, hGH,NIH-HS 20I9G, 2.2IU/mg) for RRA-GH ¡
tl25rlioao-hcn eras prepared by the lactopeS¡jd¿ss
enzl'me method of ThoreII and Johannson (1971) , using I mCi of
tul25t(New England Nuclear), 5 ug of hcH, 5 ug of ractoperoxi-
d.asef 5 ul of 30? hydrogen peroxide at I I I5000cli1ution, and
25 tul of 0.05M phosphate buffer, pH7.4 in a final volunre of
85 uI. At the end of one mínut,e chemical- reaction, I to 2 nl
of 0.025 M Tris-ItCl , pH 7.6 was added irnmedíateLy to the
reaction tùbe, after 5 ul of the reaction mixture r as taken
out for specific activity determination. Unreacted iodide and
damaged honnone r,lrere separated from intact iodinated hormone
by 9e1 filtration on Sephadex G-100 colunn (I.5 X 50 c¡n) using
0.025 M Tris-HCI ,pH 7.6 as elutÌng buffer. The Sephadex G-IOO
column was pre-treated at once with l-2 tnl of 0.025M Tris-HCl .
pH 7.6 containíng 2.5* bovine serum albumin (BSA) in /v in
order to ¡ninimize the loss of iodinated proteins.
ln order to deter¡nine the specific activity of iodin-
ated hormone, S ut of the reaction nixture was removed and
diluted brÍth l ml of Trisr.Hcl buffer, pH 2.6 containing no
BSA. Then 0.1- ml of 0.01 M phosphate buffered saline, pH ?.4
containing 0.18 BSA and 2 mI of 108 trichloroacetic acid