Biology Reference
In-Depth Information
removing the acidic subunit of beta-conglycinin from total soybean
(
G. max
(L.) Merr.) seed proteins [
55
] (Fig.
2c
). If suitable anti-
bodies are available [
55
], then immunoremoval can be a useful
strategy for depletion of SSP (Fig.
2d
). Application of any of these
methods will need to be individually refi ned. For example, antibod-
ies and lectins might need to be chemically cross-linked in order to
be useful if SSP are prepared under denaturing conditions [
58
].
In principle, it should be possible to deplete a sample of SSP by
either differential solubility or isoelectric precipitation (Table
1
).
In fact, these simple strategies seldom work satisfactorily. For
example, when a total protein fraction prepared from developing
soybean seeds was dialyzed exhaustively against deionized water it
should have yielded a globulin-depleted albumin fraction and an
insoluble globulin fraction. Both fractions, however, were heavily
cross-contaminated (JAM, unpublished). Results were better with
rice seed glutelins, and best with maize prolamins (Fig.
4a
).
Extraction of dry, mature maize endosperm did yield a prolamin
(zein) enriched fraction, but some of the zeins remained associated
with the ethanol-insoluble material. This might be because of the
oxidative cross-linking of the zein subunits [
59
]. If this were the
case then results might have been improved by including 2 %
(V/V) 2-mercaptoethanol in the 70 % ethanol solvent.
The fi rst practical, usable, and commercially available device
for Liquid Phase-IEF/isoelectric precipitation was dubbed the
Bio-Rad Rotofor. This method has been used extensively in both
shotgun proteomic analyses [
60
] and in individual, specifi c applica-
tions [
61
]. More recently, Agilent Technologies has marketed a
LP-IEF system called the 3100 OFFGEL Fractionator. In this sys-
tem, the pH gradient results from use of an immobilized pH gradi-
ent strip, rather than pH limit ampholytes. The Rotofor and
OFFGEL, and similar devices such as the ZOOM IEF Fractionator
[
62
] are capable of suitable SSP depletion. The disadvantages of
include being technically diffi cult and expensive.
Krishnan and associates [
57
,
63
] adopted a somewhat different
strategy for depleting total soybean seed proteins of the very abun-
dant 7S and 11S globulins. They found that incubation of a total
protein fraction with 10 mM Ca
2+
led to precipitation of the seed
globulins glycinin and
-conglycinin (Fig.
4b
). This simple and
inexpensive method removed 87 ± 4 % of the SSP from the sample
and allowed identifi cation of 541 previously inconspicuous pro-
teins. In preliminary experiments, this method appeared to work
equally well with seeds from other legumes (e.g., peanut, bean,
pea, and alfalfa). While it would be diffi cult to improve the ease or
cost of the calcium-precipitation strategy, it does not always work
perfectly and might be more productively combined with one of
the other methods described herein.
Many of the abundant SSP are N-glycosylated, making lectin-
affi nity chromatography a potentially useful depletion method in
this context [
64
,
65
]. Concanavalin A (con A) recognizes and binds
β
