Published online January 22, 2007
Rapid Assessment of Glutenin and Gliadin in Wheat by UV Spectrophotometer
J. Suchy, O. M. Lukow,* D. Brown, R. DePauw, S. Fox, and G. Humphreys
ABSTRACT
Traditional breeding of common wheat (Triticum aestivum L.) con-
centrates largely on the improvement of protein quality because of the
importance of protein in end-product functionality, nutritional value,
and economic impact. New, rapid, and inexpensive protein quality
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
tests are required to identify premium quality families from large and
diverse early-generation breeding populations. In this study, a simple
method was designed using organic solvents to divide wheat proteins
into monomeric-rich (single-chain, mostly gliadin), polymeric-rich
(multichain, mostly low- and high-molecular weight glutenin), and
total soluble protein (monomeric and polymeric protein). Monomeric-
rich and total soluble protein fractions were quantified at 280 nm with
an ultraviolet (UV) spectrophotometer. Protein fractions were ex-
pressed in terms of absolute concentration and as a proportion of total
soluble protein. A strong linear relationship between the protein con-
centration in the fractions and the absorbance reading indicated that
the method could accommodate a large range of protein fraction con-
centrations. The specific relationships between quantity and propor-
tion of monomeric/polymeric protein fractions and dough quality tests
allowed for the design of an algorithm eliminating poor quality lines
from further breeding assessment. Simplicity, reliability, low cost, and
a potential for automation could make this UV-spectrophotometric
method suitable for routine use in wheat breeding programs.
I T HAS BEEN long recognized that the amount of wheat
protein and its functional quality determine the prop-
erties of dough and breads (MacRitchie and Lafiandra,
1997). Fractionation and characterization of wheat pro-
tein has identified specific protein constituents that are
associated with functional breadmaking quality. It is the
gliadin and glutenin proteins that confer dough function-
ality. Monomeric wheat proteins, single chain proteins
consisting mainly of gliadins (storage protein), are asso-
ciated with increased viscosity and extensibility of dough
(Uthayakumaran et al., 1999; Uthayakumaran and Lukow,
2005). Polymeric proteins, multichain proteins comprised
mainly of glutenin, are related to dough strength as mea-
sured by extensigraph maximum resistance (MacRitchie,
1999) and by farinograph dough development time and
mixograph work input (Sapirstein and Fu, 1998).
In light of the strong relationship between wheat pro-
tein fractions and dough functional behavior, there have
been efforts to establish reliable protocols for quantita-
tive measurement of the monomeric and polymeric pro-
J. Suchy, O.M. Lukow, D. Brown, S. Fox and G. Humphreys, Agric. and
Agri-Food Canada, Cereal Research Centre, 195 Dafoe Rd., Winnipeg,
MB, Canada R3T 2M9; R. DePauw, Agric. and Agri-Food Canada,
Semiarid Prairie Agricultural Research Centre, 1 Airport Rd., Swift
Current, SK, Canada S9H 3X2. Publication No. 1931 of Cereal Re-
search Centre, Winnipeg, MB. R3T 2M9, Canada. Received 25 May
2006. *Corresponding author (olukow@agr.gc.ca).
Published in Crop Sci. 47:91–99 (2007).
Seed Physiology, Production & Technology
doi:10.2135/cropsci2006.05.0344
ª Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
91
teins in wheat. Among many sophisticated methods such
as sodium dodecyl sulfate- and acid-polyacrylamide gel
electrophoresis, capillary electrophoresis, and reverse
phase- and size exclusion-high performance liquid chro-
matography (HPLC), the latter method has become
the more common tool in identifying and quantifying
wheat protein constituents (Huebner and Bietz, 1999).
The complexity of the method and high costs associated
with purchasing and maintaining HPLC equipment,
however, limit its routine use by wheat breeders.
A number of methods for fractionating wheat pro-
teins have been developed, starting with the original
fractionation procedure proposed by Osborne (1907).
Although significant progress in advancing these meth-
ods has been made, most methods rely on the differen-
tial solubility of wheat proteins in a variety of solvent
systems and require quantitative measurement of each
fraction by either the Kjeldahl or nitrogen combustion
(Dumas) methods.
Some protein fractionation schemes were developed
to allow characterization of a number of wheat proteins
on a microscale (Sapirstein and Fu, 1998; Fu and Kovacs,
1999). Sapirstein and Fu (1998) proposed a proce-
dure based on the differential solubility of protein from
100 mg of flour involving sequential extraction with 50%
(v/v) propan-1-ol to obtain a gliadin-enriched fraction
called the 50PS fraction. Further extraction of the
residue with 50% propan-1-ol and 1% dithiothreitol
(DTT) yielded a mixture of high molecular weight
(HMW-GS) and low molecular weight (LMW-GS) glu-
tenin subunits called the 50PI fraction. Upon increasing
concentration of propan-1-ol to 70% in the 50PS frac-
tion, Sapirstein and Fu (1998) were able to separate and
quantify the amount of monomeric and polymeric pro-
tein in wheat flour.
A simpler separation of monomeric protein from glu-
tenin in a one-step procedure was proposed by Fu and
Kovacs (1999) using a combination of NaI and propan-
1-ol. A routine fractionation, developed by Suchy et al.
(2003), was based on the one-step separation of mono-
meric protein from glutenin and sequential extraction of
Abbreviations: 50PS, gliadin-rich; 50PI, glutenin-rich; AU, absorbance
unit; BWE, mixograph band width energy; DDT, farinograph dough
development time; ETP, mixograph energy to peak; FAB, farinograph
absorption; FPC, flour protein content; HMW-GS, high molecular
weight glutenin subunits; HPLC, high-performance liquid chromato-
graphy; LFV, lower protein fraction value; LMW-GS, low molecular
weight glutenin subunits; LQV, lower quality value; LV, loaf vol-
ume; mb, moisture basis; MDT, mixograph development time; MTI,
farinograph tolerance index; NIR, near infrared reflectance; PBW,
mixograph peak band width; PKH, mixograph peak height; PP, protein
parameter; QP, quality parameter; RSD, relative standard deviation;
SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis;
STA, farinograph stability; TEG, mixograph total energy under curve;
TQS, total quality score; TS, total score; TSP, total soluble protein; UFV,
upper protein fraction value; UQV, upper quality value; UV, ultraviolet.
 92 CROP SCIENCE, VOL. 47, JANUARY–FEBRUARY 2007
the remaining residue. Sapirstein and Fu (1998) pointed
out that the measurement of purified monomeric and
polymeric proteins does not necessarily improve the cor-
relation between the proportion of fractions in flour and
mixograph dough parameters, when compared with less
purified fractions such as 50PS and 50PI, respectively.
Modifying the procedure to allow the use of nitrogen com-
bustion analysis (Bean et al., 1998; Suchy et al., 2003)
resulted in faster and safer analyses but still proved to
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
be expensive.
A solution to this problem could be in the form of a
simple, rapid, and inexpensive methodology utilizing a
UV spectrophotometer to measure the peptide bond
which absorbs strongly in the far UV region with a max-
imum at 190 nm. For practical purposes, the wavelength
range from 205 to 210 nm is used because of less in-
terference of oxygen (Aitken and Learmonth, 2000).
This method requires accurate calibration of the spec-
trophotometer in the far UV region. The presence of
many buffers and chemicals components may strongly
interfere with the radiation signal. A second approach to
quantify the protein would be to measure the absorption
of radiation in the 280-nm region (nearUV), which de-
pends primarily on the content of tyrosine and trypto-
phan and to a lesser degree on phenylalanine (Aitken
and Learmonth, 2000).
Some of the examples of utilization of radiation mea-
sured at near-UV wavelength (280 nm) were reported
by Reichardt et al. (1987) for protein in milk, Vekshin
(1988) for protein in biological suspensions, Zhang et al.
(1993) for tyrosine phosphatase, Lieske et al. (1997) for
beta-lactoglobulin, Bauer et al. (1999) for whey protein,
and Ferreira et al. (2000) for casein. However, only one
study of wheat protein was reported at near-UV wave-
length (281 nm) (Gàbor, 1989). Studies of Sapirstein and
Johnson (1996, 2000) investigated usage of far-UV
wavelength at 214 nm for quantification of wheat pro-
tein groups.
All of the above approaches utilized some sort of
calibration to measure protein content. It is difficult to
find an absolute protein calibration standard that can
work with specific wheat proteins. Gàbor (1989) and
Sapirstein and Johnson (1996, 2000) used specific wheat
protein fractions extracted from one or two varieties.
This may lead to several problems such as the lack of
universality of these calibrations to all wheat samples
and the frequent need to update calibrations because of
year-to-year or location-to-location variability.
In Canada, in order for new wheat varieties to be
registered and grown, they must conform to established
ranges of quality traits (Lukow, 1991). New varieties
must conform to highly specific kernel characteristics,
protein content, and functional quality specifications
prescribed for the targeted wheat class. Canada Western
Red Spring wheat class is hard wheat with superior
milling and baking quality, the Canada Western Extra
Strong wheat class is hard wheat with extrastrong gluten
suitable for blending purposes, and the Canada Prairie
Spring wheat class is medium hard and medium-strength
wheat suitable for hearth breads, Asian noodles, and
related products (Uthayakumaran and Lukow, 2003).
Canadian wheat breeders need to develop and imple-
ment limits of quality traits, particularly dough strength,
in their breeding programs to meet these targets. The
methodology described here is a way to increase the effi-
ciency of screening wheat lines for protein functionality.
The experiments reported here had two objectives: (i)
to develop a simple and reliable method for measuring
the quantity and proportion of wheat protein solubility
groups in flour utilizing the spectrophotometer at near-
UV wavelength and (ii) to predict wheat quality char-
acteristics by relating the quantity of specific proteins in
wheat to rheological properties.
MATERIALS AND METHODS
Six diverse quality wheat genotypes from the Canada West-
ern Red Spring class (Superb, AC Eatonia) (Townley-Smith,
2000; DePauw et al., 1994), Canada Prairie Spring Red class
(Alpha) (DePauw et al., 1991), Canada Prairie Spring White
class (AC Karma, 9127:CS7C) (Knox et al., 1995; DePauw
et al., 1998), and the Canada Western Extra Strong class
(Glenlea) (Evans et al., 1972) were used in this study. Three
doubled haploid populations were produced, Alpha/AC Ea-
tonia, Superb/9127:CS7C, and Glenlea/AC Karma, using the
maize pollination technique (Thomas et al., 1997). Seed of the
doubled haploids were multiplied in a contra season nursery
in New Zealand.
The three doubled haploid populations were grown as three
separate experiments and the parents were repeated three
times within in each experiment. A randomized lattice design
with two replications at each location was used. The exper-
iments were grown in western Canada near Swift Current, SK,
on Swinton loam soil (Orthic Brown Chernozem), Indian
Head, SK, on Indian Head heavy clay (Rego Black Cherno-
zem), and Glenlea, MB, on Red River poorly-drained clay soil.
Grain samples from the trial near Glenlea, MB, were analyzed
on an individual replicate basis, while the samples from the
trials near Swift Current, SK, and Indian Head, SK, were
analyzed as a composite of the two replications. These three
experiments generated a total of 384 samples that were de-
rived from the three crosses as follows: Alpha/AC Eatonia, 120
samples, Superb/9127:CS7C, 144 samples, and Glenlea/Karma,
120 samples. The samples were subsequently analyzed to de-
velop the methodology reported.
The evaluation of the UV-spectrophotometric method for
prediction of wheat quality was performed using breeders’ sets
of early-generation lines grown in years 2003–2004. The West-
ern Bread Wheat (WBW) A-yield tests represented F9 wheat
lines. They were grown to evaluate their adaptation to western
Canadian locations which included yield potential, optimal
maturity, acceptable straw strength and height, and resistance
to local pathogens.
Protein Fractionation and Quantitation
Duplicates of 24 flour samples [10 mg at 140 g kg21 moisture
basis (mb)] were weighed directly into 2-mL microcentrifuge
test tubes. The first 24 samples were extracted with 1.8 mL of
50% (v/v) propan-1-ol for 30 min at 258C. Each individual
sample was vortexed at maximum setting (Genie 2, Scientific
Industries Inc., Bohemia, NY) for 5 s at 0 and 30 min. In
between vortexing, samples were incubated in a temperature-
controlled Eppendorf Thermomixer (Eppendorf-Netheler,
Hamburg, Germany) at 1000 RPM and 258C. After 30 min
extraction, the test tubes were equilibrated to room temper-
ature and centrifuged at 13500 g for 2 min. Following cen-
 SUCHY ET AL.: ASSESSMENT OF GLUTENIN AND GLIADIN IN WHEAT
trifugation, the absorbance of the supernatant was determined
in duplicate at 280 nm with a Biochrom Ultrospec 1000
equipped with Biochrom Data Capture software (Biochrom
Ltd, Cambridge, UK) and blanked with 50% (v/v) propan-1-ol
solution in Quartz QS-cuvettes. This fraction, called 50PS, con-
tains most of the monomeric protein (mostly gliadin) and small
amounts of glutenin (Fig. 1).
The second set of 24 samples was extracted exactly as
the first set but using a different solvent system of 50% (v/v)
propan-1-ol and 0.2% (w/v) DTT and the extraction temper-
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
ature of 558C. The spectrophotometer was blanked with 50%
(v/v) propan-1-ol and 0.2% (w/v) DTT solution. This fraction
was called the total soluble protein (TSP) and contains 90 to
95% of all proteins present in the flour (Fig. 1). The amount
of HMW-GS and LMW-GS in the flour (50PI) was calculated
as the difference between the amount of total soluble pro-
tein (TSP) and amount of gliadin-rich protein (50PS). The
order of extraction of 50PS and TSP fraction in the blocks
was randomized.
The proportion of 50PS (% 50PS) protein in flour was ex-
pressed as the ratio of absorbance of 50PS fraction to absor-
bance of TSP at 280 nm. The proportion of 50PI (% 50PI)
protein in flour was expressed as the ratio of absorbance of
Fig. 1. Sodium dodecyl sulfate polyacrylamide gel electrophoresis
(SDS-PAGE), 12% (w/v) acrylamide: M 5 molecular marker lad-
der 10 to 220 kDa, Invitrogen, Carlsbad, CA.
93
50PI to absorbance of TSP. The ratio of 50PS to 50PI serves
as a robust index of average protein molecular weight distri-
bution in flour: the higher the ratio, the lower the average
molecular weight distribution.
Protein composition was examined by sodium dodecyl sul-
fate polyacrylamide gel electrophoresis (SDS-PAGE) with
12% (w/v) acrylamide concentration in the separating gel un-
der reducing conditions (Fig. 1) according to Cloutier et al.
(2001). Flour was first extracted with 50% propanol (50PS)
and the remaining pellet was extracted with 50% propanol and
0.2% DTT (50PI). The TSP fraction was obtained by ex-
tracting flour with 50% propanol and 0.2% DTT.
DNA Evaluation
Nucleic acid was extracted from 50PS and TSP fractions
according to the method of Sambrook et al. (1989), evaluated
with 1% (w/v) agarose gel in 0.53 Tris Borate EDTA buffer
separation and visualized under UV light. Lambda-DNA and
Lambda Hind III digest (Invitrogen, Carlsbad, CA) were used
as a standard.
Wheat Quality Evaluation
Flour protein content (FPC) was determined with the Dickey-
John Instalab 600 near-infrared (NIR) analyzer (Dickey-John
Corporation, Cornwall, ON) according to AACC Approved
Method 39–11 (AACC, 2000). The Dickey-John analyzer was
calibrated by nitrogen combustion analysis (LECO Corpora-
tion, St Joseph, MI). Mixograph parameters were determined on
Buhler-milled flour (Buhler AG, Uzwil, Switzerland) at a water
absorption of 62% according to Pon et al. (1989), using a 10-g
mixograph (K&S Tool and Die, Winnipeg, MB, Canada). Pa-
rameters of interest were mixograph dough development time
(MDT), peak height (PKH), energy to peak (ETP), peak band
width (PBW), total energy under curve (TEG), and band width
energy (BWE). The 10-g farinograph test was performed and
analyzed according to Approved Method 54–21 (AACC, 2000).
Parameters of interest were farinograph water absorption
(FAB), dough development time (DDT), stability (STA), and
mixing tolerance index (MTI). Baking quality was evaluated on
full-formula dough on the basis of 35 g of flour, mixed to peak
(Mix Time) and baked using a modified AACC straight dough
long fermentation method (AACC Approved Method 10–10b;
AACC, 2000). Loaf volume (LV) was measured with a volu-
meter (National Mfg. Co., Lincoln, NE) by rapeseed displacement.
Statistical Analyses
The repeatability of the data was checked using residual
plots and the General Linear Model procedure of the Sta-
tistical Application Systems (SAS) software (SAS Institute
Inc., Cary, NC). Data were normally distributed as tested by
the SAS Normal plot (UNIVARIATE procedure). Repeat-
ability of absorbance reading for each fraction was measured
by relative standard deviation (RSD) (Lynch, 1998). Residual
variance was estimated by the SAS MIXED procedure, where
the sample (genotype 3 field replication 3 environment) was
treated as fixed effects, and the effect of blocking (laboratory
replication 3 field replication) was treated as random effects.
The degree of a sample set diversity (test of fixed effects for
sample, Table 1), a prerequisite for fractionation procedure
development, was tested by the mixed procedure in SAS with
sample (genotype 3 field replication 3 environment) treated
as fixed effects. The least significant difference (LSD) was de-
termined by the SAS GLM procedure.
 94 CROP SCIENCE, VOL. 47, JANUARY–FEBRUARY 2007

Table 1. Statistical evaluation of the protein fractions of the three combined doubled haploid populations (n 5 384).
Absorbance 50PS† Absorbance TSP† Absorbance 50PI† Proportion 50PS‡ Proportion 50PI‡ Ratio§
AU %
Range (min–max) 0.205–0.470 0.309–0.671 0.067–0.235 59.9–83.1 16.9–40.1 1.5–4.9
Mean standard deviation¶ 0.007 0.007 0.007 1.2 1.2 0.14
Mean relative standard deviation¶ (%) 2.0 1.5 4.8 1.8 4.1 5.9
LSD 0.018 0.025 0.015 1.97 1.97 0.19
SE 0.00165 0.00226 0.00159 0.225 0.225 0.022
Residual variance 0.000095 0.000126 0.000094 2.75 2.75 0.0465
Test of fixed effects for sample# (F value) 84.8 145.1 46.1 22.6 22.6 17.5
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

† Concentration of protein fractions: 50PS 5 gliadin-rich, TSP 5 total soluble protein, 50PI 5 glutenin-rich. AU 5 absorbance. Triplicate determinations.
‡ Proportion of 50PS or 50PI fractions of TSP fraction.
§ Ratio of gliadin- to glutenin-rich fraction 5 50PS/50PI.
¶ Mean standard deviation or mean relative standard deviation from triplicate determinations averaged across all samples.
# Mixed model, fixed effects of sample (location by field replication by cultivar/line). F values are significant at P # 0.0001.

RESULTS AND DISCUSSION and Stachelberger (1978) reported comparable ratios

Protein Extracting Conditions of tyrosine to phenylalanine in gliadin and glutenin
extracts when the gliadin and glutenin fractions were
The selection of suitable protein-extracting condi- prepared similarly. From these results, the relative pro-
tions, such as the type and the concentration of a solvent, portions of aromatic amino acids such as tryptophan,
time, and temperature of the extraction, was predicated tyrosine, and phenylalanine, in the 50PS, 50PI, and TSP
by the need to extract wheat protein fractions that are fractions were assumed not to differ significantly for the
relevant to wheat quality in a single-step extraction. same wheat. In our method, the same extraction con-
Propan-1-ol was selected as an extracting solvent be- ditions and solvent composition were used for extraction
cause of its relatively high stability in a range of tem- of 50PS and TSP. This allows treating the molar ex-
peratures, low volatility, low toxicity, low cost, and low tinction coefficient, e, as fixed in the Beer-Lambert law:
random radiation at 280 nm. Combinations of different
concentrations of propan-1-ol, coupled with DTT at A 5 e c l,
suitable extraction temperatures, allow the extraction of where the concentration of protein in solution (c) will be
wheat proteins within a specific molecular weight range directly proportional to the absorbance (A) at 280 nm of
(Kruger et al., 1988; Sapirstein and Fu, 1998; Fu and
Kovacs, 1999). The concentration of propan-1-ol was
fixed at 50% (v/v), which was suitable for extracting the
majority of monomeric protein at 258C as well as the
polymeric protein when coupled with DTT at 0.2%
(w/v) at 558C. This latter solvent system was sufficient to
extract 90 to 95% of the total protein (Suchy et al., 2003)
and was relatively free of random variation in the ab-
sorbance at 280 nm. The unextractable protein (5–10%
of total protein) has been reported by Sapirstein and Fu
(1998) and Suchy et al. (2003) as a residue protein that
does not relate strongly to wheat breadmaking quality.

Effect of the Wavelength

Two protein fractions, 50PS and TSP, obtained from a
number of varieties of wheat were scanned from 200- to
400-nm wavelength, and two major peaks were identi-
fied. Figure 2 demonstrates wavelength scans obtained
for AC Karma and Glenlea wheat. The first peak, lo-
cated between 200 and 250 nm, depended on the specific
protein fraction and could not be easily identified be-
cause of either excessive absorbance (.3.0 AU) and/or
large random signal noise. The second peak was iden-
tified at 280 nm and was independent of the protein
fraction analyzed. The absence of any major random
signal noise at 280 nm made this the appropriate wave-
length for our methodology. Figure 2 shows there was
a good separation between the two different wheat
samples on the basis of their 50PS and TSP spectra at
280 nm. Fig. 2. Absorption spectra for 50PS (A) and TSP fraction (B) ob-
Wu and Dimler (1963) showed comparable ratios of tained from 10 mg flour of AC Karma (12.9% FPC) and Glenlea
tryptophan to tyrosine in gliadin and glutenin fractions, (13.2% FPC).
 SUCHY ET AL.: ASSESSMENT OF GLUTENIN AND GLIADIN IN WHEAT
protein in the fraction, since the pathway length (l) was
constant (1 cm).
The interference in the absorbance readings of 50PS
and TSP fractions by nucleic acid in the 280-nm region
of the protein extracts was investigated. The 50PS and
TSP extracts had no detectable quantities (#5ng/mL) of
nucleic acid (results not shown).
Determination of Protein Level in Fractions
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
Absorbance at 280 nm increased with increasing pro-
tein concentration for the 50PS and TSP extracted frac-
tions, as shown for the cultivar Glenlea (Fig. 3). There
was a strong (r 2 . 0.99) linear relationship between
protein concentration of both fractions and absorbance
level at 280 nm (data not shown). Only flour weights of
10 mg (approx. 0.3–1.1 mg protein/mL) would be ac-
commodated within the reliable absorbance (AU) range
0.250 to 0.750 (Aitken and Learmonth, 2000) without
need for extensive dilutions. A major reason for using
10 mg flour samples (140 g kg21 moisture) in this study
was to avoid a laborious dilution step. The lack of uni-
versal and absolute calibration samples made it difficult
to use calibration curves. In addition, calibration curves
would require readjustment for location and year-to-
year variation. Our approach was to use protein ex-
pressed as the absolute concentration (50PS, 50PI, TSP)
or as the % 50PS and % 50PI within the same sample
(extracted in the same set). For the purpose of eval-
Fig. 3. Response of absorbance to increasing protein concentration for
50PS (A) and TSP fraction (B), extracted from different amounts
of Glenlea flour (13.2% FPC).
95
uating breeders’ wheat lines, it would be important to
compare the amount and proportions of protein frac-
tions in test wheat lines to that of standard check cul-
tivars grown under identical conditions.
Repeatability of the Method
The test for mixed effects (mixed model) indicated
that the sample set (combined doubled haploid lines and
parental cultivars) had a sufficient degree of diversity in
the concentration and proportions of protein fractions to
be used for method development (Table 1). The mea-
sured day-to-day reproducibility on the same sample
was less than 2% RSD. In our controlled experiment
(n 5 384, 6.4–18.1% FPC), the mean standard deviation
remained the same (0.007) for all 50PS, TSP, and 50PI,
but the variance associated with the error of the day-to-
day reproducibility for different wheat samples was
dependent on the magnitude of each protein fraction
measured and ranged from 1.5 to 4.8% mean RSD
(Table 1).
The effect of laboratory replication and experimental
blocking (random effects) did not have a significant ef-
fect on the variation in the absolute amounts and pro-
portions of protein fractions (results not shown).
In the routine screening of breeders’ lines, duplicate
absorbance readings would be accepted if the absolute
difference between two readings R1 and R2:
|R1 2 R2 | # tc 3 (2 3 Residual Variance)0:5
where tc is two sided critical value of t » 2 at df . 30 and
a/2 5 0.025 and where the residual variance for specific
protein is stated in Table 1. For example, two replicate
readings of TSP protein absorbance (0.563 and 0.585)
would be accepted, since the absolute difference
between two readings (0.022) is lower than [tc 3 (2 3
Residual Variance)0.5] 5 0.032.
The Relationship between the Proportion of
Protein Solubility Groups and Dough Quality
The success of the UV-spectrophotometric method-
ology depends on the level and year-to-year stability of
the relationship between the amount of extracted pro-
tein fractions and the dough quality parameters. Table 2
summarizes some of the relationships found between
the protein fractions determined by the UV-spectropho-
tometric method and dough quality parameters ob-
tained for the three combined doubled haploid wheat
populations. The amount of protein fractions correlated
better to the mixograph (MDT, PKH, PBW, and TEG),
farinograph (DDT), and baking tests (LV) as compared
with protein fractions expressed as the proportion of
TSP. However, % 50PI correlated better to mixograph
energy to peak (ETP) and the farinograph (STA and
MTI) than the absolute amounts of protein fractions.
The direction of the relationship (positive or negative cor-
relations) between protein fractions and dough quality
parameters becomes important when predictions of
dough quality are attempted on the basis of the amount
and proportions of protein fractions (Table 3).
 96 CROP SCIENCE, VOL. 47, JANUARY–FEBRUARY 2007

Table 2. Pearson correlation coefficients (r) between dough quality be differentiated by calculated LSD. When the mean
parameters and protein fractions of the three combined doubled amounts of fractions (50PS, 50PI, and TSP) or propor-
haploid populations (n 5 384) determined by the UV spectropho-
tometer at 280 nm. tion of fractions (% 50PS, % 50PI) between two wheat
samples is equal or greater than the LSD value (a 5
Protein concentration† Proportion of TSP‡
0.05) (Table 1), there is 95% confidence that two wheats
Test 50PS 50PI TSP % 50PS % 50PI are different in the amount or proportion of protein
Mixograph§ MDT 20.69 NS¶ 20.51 20.35 0.35 fractions in the flour.
PKH 0.73 0.68 0.82 20.27 0.27 The typical course of development of a wheat cultivar
ETP 20.32 0.35 20.04 20.61 0.61
PWB 0.70 0.66 0.79 20.27 0.27 in western Canada from initial cross to the registration
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

TEG 0.64 0.74 0.79 20.39 0.39 process has been described in detail by Lukow (1991).
BWE 0.39 0.75 0.63 20.56 0.56 Because of a large number of experimental lines, un-
Farinograph# FAB 0.63 0.49 0.66 NS NS
DDT 0.38 0.70 0.59 20.51 0.51 suitable lines will be eliminated from further breeding
STA NS 0.47 0.20 20.54 0.54 at every successive level of the cultivar development.
MTI NS 20.30 NS 0.44 20.44
Bake†† LV 0.63 0.66 0.75 20.33 0.33
The decision to remove a particular experimental line is
Mix time 20.56 0.05 20.35 20.42 0.42 based on many criteria such as agronomic performance,
FPC‡‡ 0.85 0.69 0.91 20.21 0.21 disease resistance, and quality attributes (Lukow, 1991).
† Concentration (AU) of protein fractions: 50PS 5 gliadin-rich, 50PI 5 Most sets of lines to be evaluated contain cultivar
glutenin-rich, TSP 5 total soluble protein. checks that define lower- and upper-level quality values.
‡ Proportion of 50PS or 50PI fractions of TSP fraction, %.
§ Mixograph parameters: MDT 5 mixograph development time, min; PKH In the process of screening wheat samples, the level of
5 peak height, %torque; ETP 5 energy to peak, min* %torque; PWB 5 each of the protein parameters is compared with the
peak band width, %torque; TEG 5 total energy under curve, min* lower and upper levels of the checks. The schematic pro-
%torque; BWE 5 band width energy, min* %torque.
¶ Not significant at P # 0.05. cess of scoring lines is depicted in Fig. 4A. For example,
# Farinograph parameters: FAB 5 water absorption, %; DDT 5 dough for 50PS, exceeding the upper-value will generate one
development time, min; STA 5 stability, min; MTI 5 mixing tolerance
index, BU.
negative score (demerit), whereas falling below the
†† Bake test parameter: LV 5 loaf volume, cm3; Mix Time 5 time to mix lower value will generate one positive score (merit). A
to peak. high 50PS value indicates that there is excessive gliadin
‡‡ Flour protein content, %.
in the wheat, resulting in weak dough as characterized
by low MDT, ETP, DDT, STA, and high MTI (Table 3).
Suitability of UV-Spectrophotometric Method for Accumulation of two or more demerits indicates that the
Early-Generation Screening sample might have excessively weak dough character-
An effective differentiation between wheat lines vary- istics. Similarly, accumulation of two or more merits in-
ing in dough quality, based on the amount and pro- dicates that the sample might have overly strong dough
portion of protein solubility groups in flour or whole characteristics for this particular wheat set.
grain, was important in the development of this method. The direction of the score (negative or positive) will
Although a wide range in the absolute amounts and pro- depend on the specific protein parameter and is outlined
portion of proteins has been observed for the combined in Table 3 derived from Table 2. The previously pub-
doubled haploid set (Table 1), individual samples can lished results of Sapirstein and Fu (1998) and Suchy

Table 3. Relationship between protein fractions determined by the UV spectrophotometer at 280 nm and dough quality of the three com-
bined doubled haploid populations (n 5 384).
Effect on dough quality†
Protein fraction
parameter Protein composition At high level At low level
50PS‡ Mostly monomeric protein (gliadin) Excessive gliadin Y weaker dough (low MDT, Insufficient gliadin Y stronger dough
and a small amount of glutenin. ETP, STA, mix time, and high MTI) (high MDT, ETP, STA, mix time, and
low MTI)
50PI‡ Mostly polymeric protein (glutenin). Excessive glutenin Y stronger dough (high Insufficient glutenin Y weaker dough
MDT, ETP, STA, mix time, and low MTI) (low MDT, ETP, STA, mix time, and
high MTI)
TSP‡ Total soluble monomeric and Overall concentration of protein too high Y Overall concentration of protein too
polymeric protein. stronger dough might occur at lower low Y weaker dough might occur at
level of absorbance (high MDT, ETP, lower level of absorbance (low MDT,
STA, mix time, and low MTI) ETP, STA, mix time, and high MTI)
% 50PS§ Proportion of gliadin-rich protein Excessive gliadin Y weaker dough (low MDT, Insufficient gliadin Y stronger dough
of total soluble protein. ETP, STA, mix time, and high MTI) (high MDT, ETP, STA, mix time, and
low MTI)
% 50PI§ Proportion of glutenin-rich protein Excessive glutenin Y stronger dough (high Insufficient glutenin Y weaker dough
of total soluble protein. MDT, ETP, STA, mix time, and low MTI) (low MDT, ETP, STA, and high MTI)
Ratio¶ Proportion of gliadin-rich protein Excessive gliadin Y weaker dough (low MDT, Insufficient gliadin Y stronger dough
of glutenin-rich protein. ETP, STA, mix time, and high MTI) (high MDT, ETP, STA, mix time, and
low MTI)
† Dough parameters: MDT 5 mixograph development time, min; ETP 5 mixograph energy to peak, min
% torque; DDT 5 farinograph dough development
time, min; STA 5 farinograph stability, min; MTI 5 farinograph mixing tolerance index, BU; Mix Time 5 time to mix the dough to peak in bake test, min.
‡ Concentration (AU) of protein fractions: 50PS 5 gliadin-rich, 50PI 5 glutenin-rich, TSP 5 total soluble protein.
§ Proportion of 50PS or 50PI fractions of TSP fraction, %.
¶ Ratio of gliadin- to glutenin-rich fraction, 50PS/50PI.
 SUCHY ET AL.: ASSESSMENT OF GLUTENIN AND GLIADIN IN WHEAT 97
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.

Fig. 4. Flowchart diagram of wheat scoring based on the wheat protein fractions quantified by the UV spectrophotometer (A) and by standard
quality evaluation (B).

et al. (2003) also support the general conclusions on
dough behavior given in Table 3. The decision of as-
signing a positive or negative score to a wheat sample
will depend on whether the parameter under evaluation
is 50PS, % 50PS, Ratio (50PS/50PI), or MTI. According
to Fig. 4A, if the protein parameter meets the first cri-
teria of being less than the lower fraction value, there is
progression to the second decision stage. If the pa-
rameter is 50PS, % 50PS or Ratio, the sample is given a
score of 11; otherwise, the score is 21.
Several breeders’ WBW A-yield tests for Canada
Western Red Spring wheat grown in western Canada in
2003 and 2004 were evaluated by the proposed UV-
spectrophotometric procedure. Protein fractions were
 98 CROP SCIENCE, VOL. 47, JANUARY–FEBRUARY 2007
extracted in duplicate in a randomized design. Individ-
ual samples were scored according to Fig. 4A on the
basis of protein fraction parameters and results are
summarized in Table 4. The same samples were also
scored for dough quality according to the mixograph
(MDT, ETP), farinograph (DDT, STA, and MTI), and
bake test (LV, Mix Time) as outlined in Fig. 4B. Accu-
mulation of two or more negative scores for the same
sample indicated that the sample had weak dough char-
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
acteristics. Two or more positive scores indicated ex-
cessively strong dough mixing strength characteristics
for the particular breeding test (Table 4).
The measure of the successful prediction of dough
quality was defined as the number of samples with
correctly identified quality deficiencies by the UV-
spectrophotometric method to the number of samples
identified to have quality deficiencies by standard dough
quality tests (Table 4). By this definition, the majority of
samples belonging to WBW A-test were successfully
predicted, that is, 22/25 samples or 88%. The remaining
12% would be classified as incorrect predictions and the
consequence would be elimination of the samples from
the breeding population if no other quality data was
considered. If an overly strong or weak type of wheat
line is not identified by the UV-spectrophotometric
method, it will be identified in subsequent advanced
quality tests.
Although the presented methodology uses upper and
lower check values (two-sided), it can be easily modified
to use only a lower check value (one-sided) to eliminate
wheats with weak dough strength characteristics. Sim-
ilarly, using only an upper check value could be used to
eliminate wheats with very strong dough characteristics.
The decision whether to use two-sided or one-sided
control check values will depend on the aim of each
individual breeding program. The application of the
UV-spectrophotometric method is most suitable for
early-generation wheat screening (F4–F8), where the
differences in quality attributes may be large, and there
are large numbers of small-sized lines.
Potential for Automation
Although the UV-spectrophotometric procedure is
rapid compared with other methods, there is a quanti-
Table 4. Comparison of UV-spectrophotometric protein prediction and dough quality evaluation of advanced wheat yield tests.
Dough quality characteristics†
Test name No. of samples No. of check cultivars Too weak
WBW A-3¶, 2003 4 4
WBW A-4, 2003 11 4 3
WBW A-1, 2004 9 5 1 1
WBW A-2, 2004 12 5 1
WBW A-3, 2004 22 5 4
WBW A-4, 2004 18 5 4
† Measured by: MDT 5 mixograph development time, min; ETP 5 mixograph energy to peak, min
% torque; DDT 5 farinograph dough development time,
min; STA 5 farinograph stability, min; MTI 5 farinograph mixing tolerance index, BU; LV 5 loaf volume, cm3; and Mix Time 5 time to mix the dough to
peak in bake test, min.
‡ UV-spectrophotometric method based on 10-mg sample size.
§ Proportion of the samples predicted to have quality deficiencies by the UV-spectrophotometric method to the number of samples identified to have quality
deficiencies by standard dough quality tests.
¶ WBW 5 Western Bread Wheat A-yield tests.
tative limit on the number of samples that one operator
can process in a single day (approx. 72 samples per day).
The most time consuming activities in this procedure are
sample weighing and the measurement of absorbance.
The application of advanced UV spectrophotome-
ters with robotic capabilities could greatly increase the
throughput and reliability of the method. Preliminary
experiments in our laboratory with a modified Breeze
HPLC system (Waters Corp., Milford, MA) acting as a
robotic station, indicated that the throughput of this
method would nearly double (results not shown). Fur-
thermore, because of the ability to make dilutions with
reliable automatic injector and washing cycle, the
method could accommodate larger flour/meal sample
size and further decrease the associated error. The av-
erage coefficient of variation values for duplicate ran-
domized samples were observed to be well below 1%,
measured either as the height or as the area of the re-
sponse. Another potential for automation is the process
of scoring the samples, which could be performed by
specially designed software according to algorithm out-
lined in Fig. 4A.
CONCLUSIONS
The pressure to develop less expensive and faster proto-
cols for quality evaluation is an ongoing issue for wheat
breeders. The introduction of physicochemical tests that
indirectly measure wheat quality traits are a viable alter-
native to standard dough quality and end-product testing.
The challenge is to develop a single test that will predict a
wide range of dough and end-product characteristics.
The proposed UV-spectrophotometric method for the
extraction and quantitation of major wheat protein sol-
ubility groups has the potential to be used as a rapid,
robust, and inexpensive procedure to screen early-
generation wheat lines for dough strength. There is gen-
erally a direct relationship between the amount and
quality of various wheat protein fractions and the dough
rheological properties that define dough strength. Cap-
italizing on the latter relationship, an algorithm for pre-
diction of dough strength in early-generation wheat lines
was developed and successfully assessed against standard
rheological quality evaluation. The experimental wheat
UV-spectrophotometric prediction‡
No. of samples
Too strong Too weak Too strong Successful prediction§
1 1 0/1
1 4 2 3/4
1/1
5 1 5 5/6
1 4 2 5/5
4 4 6 8/8
 SUCHY ET AL.: ASSESSMENT OF GLUTENIN AND GLIADIN IN WHEAT
lines were scored by comparing them with appropriate
quality benchmarks and grouping them into categories
characterized by deficient-, acceptable-, or excessive-
dough strength. This method will aid wheat breeders in
making selections of suitable lines from early-generation
wheat lines (F4 to F8) in a timely and economical manner.
The advantage of this test is the ability to process a large
number of samples and to make selections in a shorter
time and at lower cost than it would occur by standard
Reproduced from Crop Science. Published by Crop Science Society of America. All copyrights reserved.
quality evaluation using laborious dough mixing tests
such the mixograph and farinograph.
ACKNOWLEDGMENTS
Financial support by the Western Grains Research Foun-
dation and the Agriculture and Agri-Food Canada Matching
Investment Initiative is acknowledged. Technical assistance
by K. Adams and the Wheat Quality Research Team, DNA
analysis by Dr. S. Cloutier and A. Walichnowski, and statistical
assistance by Dr. S. Woods are gratefully appreciated.
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