Download Fractionation of rice glutelin polypeptides using gel filtration

Fractionation
of Rice Glutelin Polypeptides
Filtration Chromatography
S.D. SNOW
and JR
ABSTRACT
Preparative gel filtration chromatography using SepharoseCL-6B was
utilized to obtain working quantities of dissociated rice glutelin polypeptides. Rice flour samples were extracted and fractionated using
dilute Tris buffer containing either 0.5% SDS, 6M urea or 8M urea.
Gel filtration under each set of extraction conditions yielded fractions
containing dissociatedacidic and basic glutelin polypeptides, but acidic
polypeptides were more effectively separatedfrom basic polypcptides
in urea than in SDS. The presenceof glutelin polypeptides in higher
molecular weight aggregatesunder each set of buffer conditions indicated that the dissociation of these components during extraction
was not complete.
INTRODUCTION
GLUTELIN, the major rice storage protein, comprises approximately 80% of the milled rice endosperm protein (Juliano, 1972). Glutelin subunits have an approximate molecular
weight of 60 kilodaltons (KD) and consist of a heterogeneous
collection of disulfide linked polypeptides (Yamagata et al.,
1982: Zhao et al., 1983). Upon reduction and under denaturing
conditions, glutelin subunits can be dissociated into two major
fractions, i.e. the acidic or or-polypeptides and the basic or ppolypeptides. The acidic and basic polypeptides have isoelectric points betwen pH 6.5-7.5 and pH 9.4-10.3 and molecular
weights ranging from 28.5-39KD and 20-23KD, respectively (Juliano and Boulter, 1976; Villareal and Juliano, 1978;
Yamagata et al., 1982; Luthe, 1983; Zhao et al., 1983; Robert
et al., 1985; Wen and L&he, 1985). Glutelin shares a number
of similarities with the principal, salt-soluble globulins of oat,
pea, and soybean including subunit biosynthesis, molecular
weight, amino acid composition and immunological properties
(Zhao et al., 1983; Robert et al., 1985; Wen and Luthe, 1985;
Takaiwa et al., 1986). Despite these similarities with the ‘legumin-like’ proteins, post-translational modification of the saltsoluble precursor polypeptides results in mature subunits that
are largely insoluble in salt solutions and classified as glutelins
(Yamagata et al., 1982). The reasons for this insolubility are
unknown, although its low salt-solubility has been attributed
to such factors as extensive subunit aggregation (Palmiano et
al., 1968; Robert et al., 1985; Sugimoto et al., 1986) and
glycosylation (Wen and Luthe, 1985). Current efforts are directed toward a more thorough characterization of the rice glutelin polypeptides and the reasons for insolubility. Glutelin
polypeptides, extracted under denaturing and reducing conditions, are typically fractionated using ion exchange chromatography or chromatofocusing(Juliano and Boulter, 1976; Zhao
et al., 1983; Wen and Luthe, 1985; Sarker et al., 1986). These
studies, however, do not consider the degree of polypeptide
dissociation following extraction. Preparative sodium dodecyl
sulfate polyacrylamide gel electrophoresis (SDS-PAGE) provides dissociated polypeptide samples (Krishnan and Okita,
Both authors were with the Dept. of Food Science, Univ. of
Arkansas, Route 7 I, Fayetteville, AR 72703. Author Snow’s present address is Molecular Probes Inc., Eugene, OR 97402. Author
Brooks is now with Product Research &Development,
Ross Laboratories, 625 Cleveland Ave., Columbus, OH 43216. Address
inquiries to Dr. Brooks.
730-JOURNAL
OF FOOD SCIENCE-Volume
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using Gel
BROOKS
1986), but the method is tedious and sample quantities are
small. In addition, removal of SDS following fractionation is
difficult and could interfere with further efforts to characterize
the polypeptides. Attempts to fractionate glutelin polypeptides
using gel filtration chromatography appear promising but have
met with limited success(Juliano and Boulter, 1976; Villareal
and Juliano, 1978; Chen and Cheng, 1986). Fractions containing dissociated acidic and basic polypeptides can be recovered but are found to be poorly separatedfrom one another
even when column lengths are extended to 100 cm. Further,
because each of these studies use sample buffers containing
0.5% SDS, the difficulties associatedwith the removal of the
detergent from the samples still remain.
In contrast to SDS, urea has little effect on methods to further characterize or fractionate the polypeptides, including isoelectric focusing, ion exchange and chromatofocusing. If
necessary, urea can be readily removed from sample preparations using dialysis or desalting columns. This study was
conducted to further evaluate the use of preparative gel filtration chromatography for obtaining working quantities of dissociated acidic and basic rice glutelin polypeptides. Samples
BP
PP
Fig. 1 -SDS-PAGE patterns for glutelin extracts: (lane 1) BioRad low molecular weight standards (in kilodaltons);
(lane 2)
6M urea extract, 15ug protein; (lane 3) 8M urea extract, 15p.g
protein; (lane 4) 0.5% SDS extract, 15pg protein. The letters AP
and BP refer to the acidic and basic polypeptides
of glutelin,
respectively. PP refers to the prolemin polypeptide.
were extracted and fractionated in 6M and 8M urea and compared with those extracted in 0.5% SDS.
MATERIALS
& METHODS
General methods
Reagents and molecular weight standards for electrophorcsis were
purchased from Bio-Rad Laboratories, Richmond, CA., and gel filtration media was obtained from Pharmacia Fine Chemicals (Piscataway, NJ). Molecular weight standardsfor gel filtration chromatography
and all other chemicals and reagents were purchased from Sigma
Chemical Company (St. Louis, MO). Dialysis tubing was Spectropor
1 with a 32mm flat diameter and 6-8KD molecular weight cutoff
(Fisher Scientific, Dallas, TX).
Short grain rice (var. Nortai) was obtained from the 1984 crop at
the University of Arkansas Experiment Station. Prior to use, it was
dehullcd, milled, and ground to a fine flour (200 mesh). Next, the
flour was defatted by stirring with ten volumes of petroleum ether for
6 hrs at 21°C decantedand allowed to air dry overnight. The defattcd
flour was stored at -20°C.
Protein contents were assayedusing the Coomassie Blue dye-binding method (Anonymous, 1976) for samples containing urea or the
Biuret method (Gornall et al., 1949) for samples containing SDS.
Bovine serum albumin was used as the reference protein.
The standard buffer used throughout this study was 0.01M TrisHCI adjusted to pH 8.0 and contained 0.05% sodium azide as a preservative. Immediately prior to use, O.OOlM dithioerythritol (DTE)
and 0.0002M phenylmethylsulfonyl fluoride (PMSF) were added as a
disulfidc bond reducing agent and protease inhibitor, respectively.
Crude glutelin extraction
6$ik
2SjkI
Defatted rice flour was extracted with ten volumes (w/v) (this volume was adequate in removal of water-and salt-soluble proteins) of
standard buffer containing 0.4M NaCl for 1 hr at room temperature
(21°C) to solubilize salt-soluble albumins and globulins. Following
centrifugation at 35,000 x g for 20 min at 20°C the supernatantwas
discarded. The residue was reextracted using standard buffer containing either 0.5% SDS, 6M urea or 8M urea for 3 hr at 21°C. The flourto-solvent ratios (w/v) used for these extractions were l:lO, 1:15, and
1:60, respectively. A crude glutelin supernatantwas obtained following centrifugation as above. Each sample was assayedfor protein and
immediately applied to the gel filtration column.
I
Gel filtration
tube number
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chromatography
Twenty-four mL aliquots of each crude glutelin solution were fractionated on a 2.5cm i.d. x IlOcm Sepharose CL-6B gel filtration
column (Pharmacia Fine Chemicals, Piscataway, NJ) previously equilibrated with the standard buffer containing either 0.5% SDS, 6M
urea or 8M urea. The column was cluted at a flow rate of 13 mL/hr,
and absorbancewas continuously monitored at 280 nm using a Type
6 optical head connected to a UA-5 Absorbance/Fluorescencemonitor
(Instrumentation Specialities Co., Lincoln, NE). Effluent was collected in 4 mL portions using a Frac-100 fraction collector (Pharmacia
Fine Chemicals, Piscataway, NJ). Column effluent for each fraction
was combined and concentratedby ultrafiltration using a 50 mL stirred
cell equipped with a YM-10 membrane (Amicon Corp., Danvers,
MA) prior to examination by SDS-PAGE. Elution volumes were detcrmined to the mid-point of each fraction. They were compared to
the elution volumes of bovine serum albumin (66 KD) and carbonic
anhydrase (29 KD) standards, which were solubilized and passed individually through the column under similar buffer conditions.
.4----
o.io*
procedure
is
Fig. Z-Sepharose
CL-GB elution patterns for glutelin
in (A] 0.5% SDS, (8) 6M urea and (C) % M urea.
65
extracted
Gel electrophoresis
SDS-polyacrylamide. gel electrophoresis (SDS-PAGE) was performed in 12% or 13% separating gels with 4% stacking gels according to a modification of the Laemmli (1970) method and using the
Protean II Slab Cell (Bio-Rad Laboratories, Richmond, CA) vertical
unit. Sampleswere diluted in buffer in which the SDS concentrations
were increased to 0.2% (w/v), and O.OOlM DTE was used in place
of mercaptoethanot as the reducing agent (Brooks and Morr, 1984).
The 0.75 m m thick gels were electrophoresedat a constant current of
13 milliamps (mA) in the stacking gel and increased to 18 mA for
migration through the separating gel. Low molecular weight protein
standards(Bio-Rad Laboratories, Richmond, CA) consisting of phosphorylase B (92.5KD), bovine serum albumin (66.2KD), ovalbumin
(45KD), carbonic anhydrase (31KD), soybean trypsin inhibitor
(21.5KD), and lysozyme (14.4KD) were simultaneously electrophoresed as reference markers. Gels were stained overnight with 0.1%
Coomassie Brilliant Blue R-250 (w/v), 40% ethanol (v/v) and 10%
acetic acid (v/v) in water and destained in the same solution excluding
the dye.
RESULTS
& DISCUSSION
THE RICE FLOUR SAMPLES formed very viscous solutions
when extracted with uiea. This was due to interactions of the
Volume 54, No. 3, 1989-JOURNAL
OF FOOD SCIENCE-731
FRACTIONATION
OF RICE GLUTELIN. . .
92.5k
66.2 k
45k
AP
31k
AP
2l.5k
BP
31k
q&w
14Ak
BP
A
B
92Sk
66.2k
45k
AP
31k
BP
I
I
starch fraction with urea and was especially evident in 8M urea
extracts. As a result, increased solvent extraction ratios were
necessary when using high concentrations of urea. The total
protein yields for samples extracted in 0.5% SDS and 8M urea
were comparable with values ranging from 7.5-80 mg/g flour.
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Fig. 3-A:
SDS-PAGE patterns for a 0.5% SDS glutelin extract
fractionated using Sepharose CL-6B: (lane 1) Bio-Rad low molecular weight standards (in kilodaltons); (lane 2) fraction 1, 17~
protein; (lane 3) fraction 2, 13p.g protein; (lane 4) fraction 3,
12wg protein; (lane 5) fraction 4, 13bg protein. 8: SDS-PAGE
patterns for a 6M urea glutelin extract fractionated using Sepharose CL-6B: (lane 1) Bio-Rad low molecular weight standards (in kilodaltons); (lane 2) fraction 1, 34~ protein; (lane 3)
fraction 2, 44~ protein: [lanes 4 and 6) fraction 3, 45~ protein,
(lane 5) fraction 4, 45pg protein. C: SDS-PAGE patterns for an
8M urea glutelin extract fractionated
using Sepharose CL-6B:
(lane 1) Bio-Rad low molecular weight standards (in kilodaltons); (lane 2) fraction 2, 19pg protein; (lane 3) fraction 2a, 35kg
protein; (lane 4) fraction 3, 34~ protein; (lane 5) fraction 4,
35~ protein. The letters AP and BP refer to the acidic and basic
polypeptides of glutelin, respectively.
Yields using 6M urea were somewhat lower at approximately
40 mg protein/g flour. Because sample viscosities in 6M urea
were reduced, this extraction required only one-fourth the solvent volume used with 8M urea.
The SDS-PAGE polypeptide profiles obtained from extraction in each buffer system are shown in Fig. 1. These results
demonstrated that glutelin polypeptides were effectively solubilized under each set of extraction conditions. The acidic and
basic polypeptides of glutelin were found to have approximate
molecular weights ranging from 31.0-38.9KD and 21.123.9KD, respectively. These values were in close agreement
with those reported previously (Chen and Cheng, 1986; Krishnan and Okit?,. 1986; Sarker et al., 1986; Wen and Luthe,
1985). In addrtron, a small amount of polypeptides with molecular weights in excess of 58KD and a major band, at approximately 14KD, were also evident. The’high molecular
weight componentswere likely comprised of residual albumins
and globulins. The low molecular weight fraction was most
probably a prolamin polypeptide that is typically reported as a
principal contaminant of glutelin preparations (Krishnan and
Okita, 1986; Wen and Luthe, 1985). Sepharose CL-6B gel
filtration chromatography using each of the denaturing buffers
resulted in the separation of the crude rice protein extracts into
approximately 4 broad and incompletely resolved fractions that
eluted over a wide molecular weight range (Fig. 2A,B and C).
The elution volumes of the principal fractions from the urea
extracts were similar, indicating generally similar molecular
weight distributions. The overall elution profile obtained in the
presence of 0.5% SDS was somewhat different from those
obtained in urea extracts. This is likely due to differences in
shape effects or protein solubility resulting from extraction in
SDS versus urea.
Each fraction from Sepharose CL-6B chromatography was
recovered, concentrated and analyzed by SDS-Page (Fig. 3A,
B and C). In the 0.5% SDS extract, the basic polypeptides of
glutelin were found distributed throughout each of the fractions, and the acidic polypeptides were found primarily in fraction 3 and, to a lesser extent, fraction 4 (Fig. 3A). In the 6M
urea extract, the basic polypeptides were again found in the
higher molecular weight regions, i.e., fractions 1 and 2, as
well as in fraction 4, the smallest molecular weight region.
The acidic polypeptides were primarily in fraction 3, although
small amounts of these polypeptides were dispersedthroughout
(Fig, 3B). When the urea concentration was increased from
6M to 8M, there was an apparent decreasein the contribution
of fraction 2 combined with an increase in the region labeled
2a (Fig. 2C). This most likely indicates a possible shift toward
decreasedpolypeptide aggregation. Fractions, 2, 2a and 4 contained the basic polypeptides, and the acidics were again predominant in fraction 3 with minor amounts present in other
fractions as was found in 6M urea (Fig. 3C). In this sample,
fraction 1 yielded almost no protein upon concentration and
was omitted from SDS-PAGE.
As indicated in Fig. 2, the majority of the acidic polypeptides were recovered betwen the 66KD and 29KD molecular
weight markers under each set of extraction conditions, and a
significant portion of the basic polypeptides eluted after the
29KD marker. Many of the fractions collected for subsequent
SDS-PAGE in this study were closely spaced, and, therefore,
a certain degree of component overlap was expected. However, the elution patternsfrom gel filtration in 0.5% SDS showed
a less defined separation of the dissociated acidic polypeptides
from the basic polypeptides. These findings were in general
agreement with earlier studies (Chen and Cheng, 1986; Villareal and Juliano, 1978; Juliano and Boulter, 1976). Fractionation in 6M and 8M urea resulted in the recovery of an
enriched acidic polypeptide fraction (fraction 3) that was largely
separatedfrom the basic and prolamin polypeptides. Because
these components eluted just prior to the 29KD standard, they
appearedpresent as free polypeptides and, as such, would be
suitable for further characterization and study. Similarly, fraction 4 most likely contained free basic polypeptides, but this
fraction was heavily contaminated with the prolamin fraction
as well as acidic polypeptides. This was not surprising, because the low molecular weight cutoff for Sepharose CL-6B
was very close to that of the basic polypeptides.
The additional presenceof glutelin polypeptides eluting from
the gel filtration column at molecular weights in excess of the
66KD standard indicated that a portion of these components
was not completely dissociated by the extraction conditions
employed or was able to reaggregatefollowing solubilization.
Heating in the presence of SDS and DTT, as was done in the
preparation of samples for SDS-PAGE, provided further dissociation to free polypeptides. Although the results were not
quantitated, SDS-PAGE for each set of buffer conditions appeared to show a larger percentage of the basic polypeptides
eluting in the higher molecular weight regions (Z 66KD) than
was found for the acidic polypeptides.
Efforts to more fully characterize the dissociated glutelin
polypeptides, obtained from gel filtration chromatography in
6M urea, are currently in progress.
CONCLUSIONS
THIS STUDY demonstrated that glutelin extraction, under
denaturing and reducing conditions, did not necessarily result
in the complete dissociation of the protein into free polypeptides. The presence of heterogeneouspolypeptide aggregates
would likely confound attempts to further characterize or fractionate the individual polypeptides. Gel filtration chromatography, using Sepharose CL-6B, was effective for partially
purifying crude glutelin extracts and obtaining preparative
samplesof dissociatedglutelin polypeptides suitable for further
characterization and study. Urea was the preferred extractant,
becauseit would be less likely to interfere with further attempts
at characterization and, if required, could be readily removed.
In addition, a highly enriched fraction of dissociated acidic
polypeptides was obtained using either 6M or 8M urea. Extraction in 6M urea yielded less total protein than 8M urea,
but the overall polypeptide profiles, as indicated by SDS-PAGE,
were similar. Decreased sample viscosities during extraction
with 6M urea permitted the use of decreased solvent ratios.
This, in turn, resulted in higher protein concentrations, which
permitted a larger protein load, per fixed volume of extract,
on the gel filtration column.
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MS received 9123187; revised S/26/88; accepted 11/7/88.
Presented in part at the 47th annual meeting of the Institute of Food Technologists,
June 1987, Las Vegas, NV.
Volume 54, No. 3, 1989-JOURNAL
OF FOOD SCIENCE-733