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38 Vuayalakshmi
Table 1
Companson of Sepharose-Based Adsorbents
at Different ConcentratlonP
Lqand
cont., pM/g
dv gel
10
25
% Purity of
the final
product
98.9
95.5
Held, %
9.2
4.5
aEffect of ligand concentrahon expressed as pM
hlstidine/g gel dxy wt on the purlticahon and reld
of 1gq.
case, the lower ligand concentration (10.5 pit4 his/g dry gel) gives a
higher recovery (9.2% Ifi,) and a higher purity (98.9% pure) com-
pared to a gel with a higher ligand concentration (24.3 PM his/g dry
gel). However,. this factor may MIX with each protein studed (unpub
lished data).
5. The upstream extraction methods used to prepare the crude extract
of the protein to be purified play an important role in its retention
behavior. The following examples illustrate this.
Chymosin from calf or kid abdomen (sodium chloride extract)
was selectively retained on a histidyl-Sepharose or histidyl silica col-
umn (8) but, when the extraction was done with sodium chloride in
the presence of sodium benzoate, no specific retention could be ob
tained (unpublished data).
In the case of purification of IgGl from human placental serum,
a comparative study of the ammonium sulfate precipitation and ethyl
alcohol precipitation gave the results, shown in Fig. 3.
In the case of an ammonium sulfate extract, all the proteins in-
cluding the IgG subclasses other than IgGI, were found in the break-
through fractions (peak 1, Fig. 2). Whereas, in the case of
ethanol-precipitated extracts, the albumin was not retained (peak 1,
Fig. 3) and the IgG fractions other than IgGl were slightly retarded
(peak 2, Fig. 3) while IgG, was strongly retained and eluted with 200
mA4 sodium chloride (peak 3, Fig. 3).
The purity of the extract used for the chromatographic step is
another important factor. Table 2 shows the comparison of the puri-
fication of IgG1 from different placental extracts, namely A, S,, and
S,, which had the initial purities of 58 and 9076, respectively.