Comparison of procedures to determine protein content of developing bean seeds (Phaseolus vulgaris)
O. PAREDES-LOPEZ, 1'* F. GUEVARA-LARA, ~ M.L. SCHEVENIN-PINEDO 1 & R. MONTES-RIVERA 2 1Unidad Irapuato, CIEA-lnstitute Politdcnico NaL, Apdo: Postal 629, 36500 lrapuato, GTo., MOx&o (*author for correspondence); 2 Centro de Investigaciones Agrlcolas de El Bajlo, INIFAP, Celaya, Cto., Mdxico
Key words: beans, protein determination, Lowry, Bradford, Kjeldahl methods
Abstract. Micro-Kjeldahl, Lowry and Bradford procedures were compared for determining the protein content of Phaseolus vulgaris seeds during their development. Micro-Kjeldahl and biuret techniques were also compared with mature seeds of a normal and a genetically- improved bean cultivar. The protein contents of casein and soy protein isolate were as well estimated by these four methods. For many samples of both bean and food protein products large disagreements were found between micro-Kjeldahl and the other three procedures.
Introduction
The seed protein content of Phaseolus vulgaris is influenced by the environ- mental conditions in which plant growth and seed maturation occur, the genotype of the maternal plant and the expression of genes that regulate synthesis and accumulation of protein and nonprotein fractions in the seed [12]. Since increases in protein percentage are due to proportionate reduc- tions in the nonprotein components as well as increased accumulation of proteins, it is necessary to establish the basis for the observed changes.
Different procedures for protein content determination have been used by various groups working with Phaseolus vulgaris proteins. The micro-Kjeld- ahl method has been used by Opik [15], Pusztai and Watt [18] and Paredes- Lrpez et al. [16]; the Lowry technique by Felstad et al. [5] and Leavitt et al [7]; and the Bradford procedure in our laboratory (unpublished results). The Bradford procedure has also been used by Starogcik et al. [19] to measure the protein content of peanut extracts and peanut lectin solutions.
On the other hand, it is well known that Phaseolus vulgaris is rich in components such as tannins that may interfere with the protein content determinations and no studies are available on the comparative perfor- mance of these methods for such pruposes. The objective of this preliminary
138
study was to assess the reliability of the other procedures, as compared to the micro-Kjeldahl method, for measuring the protein content of ripening and mature seeds of Phaseolus vulgaris.
Materials and methods
Bean samples.
The cultivars used for this study (Phaseolus vulgaris L., cv. flor de mayo) were sown in plots at the experimental farm of the Centro de Investigaciones Agricolas de E1 Bajlo during the spring of 1985 and 1986. The normal cultivar, flor de mayo (FM-C), was genetically improved by plant breeding and this material is now resistant to the common mosaic virus (FM-RMC). The corresponding year of harvesting is also indicated (e.g. FM-C-86). Flor de mayo is a very important cultivar in Mexico because of its desirable sensory attributes. Green pods were sampled every other day; sampling started 19 days after flowering. Mature seeds, named Ht after harvest time, were collected 45 days after flowering. The seeds were dissected out of the pods with the help of a scalpel, sorted out by size from 3- to 13 mm-length, and freeze-dried. These green beans and mature samples were milled (UD Cyclone Sample Mill, UD Corp.; Boulder, CO) to pass a 100-US mesh sieve. The bean flours were kept in tightly covered containers at - 6 0 °C.
Reference food proteins
The commercial food protein products were casein (Sigma de Mexico, Mexico, D.F.), and soy protein isolate (Ralston Purina Co., St. Louis, MO).
Moisture content
The moisture content was determined in triplicate by drying the samples in an oven with forced air at 130°C for 1 h [1].
Preparation of seed homogenates and related samples
PBS (phosphate-buffered saline )-homogenate/non-dialysable sample Seed flour was extracted with PBS (1:25 ratio), pH 7.4, in an Ultra-Turrax homogeniser (Janke & Kunkel, Staufen, FRG) at full speed for 5 min at room temperature [5]. The homogenate was magnetically stirred for 16 h at 5 °C and then dialysed against deionised water at 5 °C until the conductivity
139
i PBs
~OMOGERISA?IO~
AGITA~IO~
.L DIALYSIS
I p~S-HOMOGEgATE] NON DIALYSABLE
I NaCi-As¢orbic Acid
HOMOGENISAT~ON
$ CENTRIFUGATION AGITATION
CSNTRIFUGATION SHPERNATANT ITCA
@ l "* CENTR~FUGATION SUP~RNATANT
N~oP~S SOLUBLE/ pBS~SOLUBLE/ .......... 1 l TCA-INSOLUBLS
TCA
CENTRIFUGATION
PRECIPITATE Suspe.sidn
Na0H
l ..................... /l TCA INSOLUBLE
Fig. 1. Protein fractionation from bean seed flour.
was equilibrated at 10#mhos/cm. Dialysis tubing with a molecular weight cut-off of 6000-8000 was used (Spectra-Por 1, Spectrum Medical Industries, Inc., Los Angeles, CA). The homogenate contains mostly soluble-globulins [4, 14] and pellets in suspension (Fig. 1).
PBS-soluble/total, PBS-soluble/non dialysable and PBS-soluble/ TCA-insoluble samples The PBS-soluble/total protein sample was obtained by centrifuging the homogenate at 40000 × g for 15 min at 5 °C and discarding the precipitate. The same supernatant was dialysed as described previously to obtain the PBS-soluble/non-dialysable sample (Fig. 1).
TCA (trichloroacetic acid) has been used to avoid or reduce the presence of nonprotein molecules that may interfere with protein content determi- nations [13, 17]. An aliquot of the supernatant containing 100-200#g of protein, by micro-Kjeldahl procedure, was made up to 1 ml with deionised water and 0.2ml of 72% TCA was added. The mixture was thoroughly shaken, centrifuged at 3000 x g for 15 min and the supernatant discarded. The TCA treatment was repeated and the protein precipitate was resuspen- ded with 1 ml of 0.1 M NaOH and used to determine protein content of the PBS-soluble/TCA-insoluble sample (Fig. 1).
The PBS-soluble/total fraction contains saline-soluble proteins plus free amino acids. After the dialysis step to remove low molecular weight com- ponents, such as free amino acids, the fraction was referred to as the PBS-soluble/non-dialysable fraction. The proteins of PBS-soluble/total sample were precipitated by TCA and then were termed PBS-soluble/TCA- insoluble fraction.
140
NaCl-ascorbate soluble/total and NaCl-ascorbate soluble/TCA-insoluble samples The NaCl-ascorbate soluble proteins (Fig. 1) represent a mixture of globulins G1 and G2, acid albumins and free amino acids [9]. The NaCI- ascorbate soluble/total proteins were extracted according to previous studies [10, 20], with minor modifications: One gram of mature bean flour was homogenised with 25 ml of 0.5 M NaC1-0.25 M ascorbic acid, pH 2.5, in an Ultra-Turrax homogeniser at full speed for 5 min at room temperature. The homogenate was magnetically stirred for 1 h at 5°C and then centrifuged at 30000 x g for 30min at 5°C. The precipitate was discarded and super- natant made up to 25 ml with the same extracting solution.
The protein sample termed NaCl-ascorbate soluble/TCA-insoluble (Fig. 1) was obtained using an aliquot of the previous supernatant and treated with TCA as above. The TCA-insoluble protein was resuspended with 1 ml of 0.1 M NaOH and used to determine protein content. This fraction should be mostly composed by globulins G1 and G2, and acid albumins [9].
Protein determination
The micro-Kheldahl procedure [2] was conducted on 10-40mg of samples. PBS and NaCl-ascorbate extracted samples were also analysed by the micro-Kjeldahl method. Protein content was computed using nitrogen con- version factors of 6.25 for both bean and soy proteins, and 6.38 for milk proteins.
Protein content determinations were also performed by Lowry [8]. Brad- ford [3] and buiret [6] methods with crystalline bovine serum albumin (Fraction V, A-4503, Sigma Chemical Co., St. Louis, MO) as standard. For the analysis of the TCA-precipitated bean proteins, this standard protein was also subjected to the same TCA treatment. The total protein solubilised in NaCl-ascorbate was not quantitated by biuret due to the ascorbate interference with this method. When this extracting agent was used the protein was TCA-precipitated before biuret determination to avoid such interference.
Results and discussion
Seed weight, moisture and protein contents during seed formation
The individual seed weight reached a maximum at 13 mm-seed length (Fig. 2); the average fresh weight of mature seeds (Ht) was significantly lower. The
141
SEED LENGTH (ram)
A
4 0 0 t-~ Ild la l Oq
ia l
- I,-- "T
m
till
Fig. 2. Changes in the moisture content, fresh weight and dry weight of Phaseolus vulgar& seeds during their development. 0 Moisture content, i Fresh weight. [] Dry weight. HT = Harvest time:
seed water content declined slightly up to a seed length of 10 mm; thereafter this content decreased at an accelerated rate.
The protein weight per dry seed, determined by micro-Kjeldahl, rose steadily during the seed formation but an opposite trend was exhibited by the protein content expressed in percentage units (Table 1), as observed previously in bean cotyledons [15]. The micro-Kjeldahl protein content of the PBS-homogenate/non-dialysable sample decreased considerably from 4-mm length to Ht samples. However, the proportion of nitrogen in this sample in relation to the total seed flour protein remained constant in the 4-, 8-, and 10-mm length seeds and increased in the mature sample. The total protein solubilised in PBS followed no given trend during seed ripening; a lack of agreement was evident when the micro-Kjeldahl data of the PBS- soluble/total protein were compared to the corresponding values produced with the Lowry and Bradford procedures. The content of this protein was remarkably tow by the latter technique.
Dialysis was used to decrease the content of low molecular weight com- ponents in the PBS samples as measured by the micro-Kjeldahl, Lowry and Bradford methods. This decrease was observed by comparing the data from the PBS-soluble/non-dialysable samples with the PBS-soluble/t0tal proteins (Table 1). However, this effect was not evident with the Bradford technique.
Tab
le 1
. P
rote
in c
on
ten
t ~ o
f P
hase
olus
vul
gari
s se
eds
du
rin
g t
hei
r d
evel
op
men
t b
to
Sam
ple
/Pro
ced
ure
S
eed
len
gth
(m
m)
4 8
10
Ht
mg
of
seed
pro
tein
per
dry
see
d c
0.6
4.8
11.5
43
.3
See
d fl
ou
r p
rote
in/t
ota
l c
43.3
+
0.
1 a/
a 29
.7
__.
1.4
b/a
31.3
-}
- 1.
8 b/
a 23
.4
_+
1.0 c
/a
PB
S-h
om
og
enat
e/n
on
-dia
lysa
ble
C
23.9
_+
0.1
"/b
16
.2
q--
0.1
b/b
16
.7
~---
0.8
b/b
14
.2
±
0.4
c/c
PB
S-s
olu
ble
/to
tal
Mic
ro-K
jeld
ahl
12.6
+
1.
6 a/
c 13
.9
±
0.2
a/c
11.2
_+
0.1
a/c
13
.0
±
0.6
a/o~
Lo
wry
9.
0 _
0.5
TM
5.5
+
0.1
d/d
6.8
_ 0.
1TM
16
.3
___
0.4
a/b
Bra
dfo
rd
1.3
___
0 c/
el
0.8
q- 0
.1 e/
g 2.
9 +
0.
1 b/
g 5.
7 q-
0.6
a/g
PB
S-s
olu
ble
/no
n-d
ialy
sab
le
Mic
ro-K
jeld
ahl
8.2
+
0.5
TM
2.3
+
0.1
d/e
4.5
+
0.1
c/el
11
.0
+
0.6
a/r
Lo
wry
2.
5 +
0.
3 c/
e 1.
0 +
0
d/rg
5.
4 +
0
b/d
13.8
+
0.
1 a/¢
d
Bra
dfo
rd
1.3
q- 0
.1 e
/el
0.6
___
0.1
d/g
3.0
-}-
0.3
b/fg
6.
1 -]
- 0.
1 a/
g
PB
S-s
olu
ble
/TC
A-i
nso
lub
le
Mic
ro-K
jeld
ahl
1.0
-}-
0.2
c/el
1.
8 __
_ 0.
1 ~/
~r
3.6
___
0 b/
tg
11.6
_
0.6
"/~r
Lo
wry
0.
7 +
0
c/r
0.8
___
0 c/
g 5.
6 +
0.
3 b/
d"
14.7
__
_ 2.
0 "/¢
Bra
dfo
rd
1.4
_+ 0
~/
~r
1.4
+
0.1
¢/fg
2.
2 _+
0
b/g
5.3
_+ 0
.4 a/
g
" A
s p
erce
nta
ge
of
dry
mat
ter
un
less
sta
ted
oth
erw
ise.
Mea
n o
f tr
ipli
cate
s _+
sta
nd
ard
dev
iati
on
. M
ean
s w
ith
th
e sa
me
lett
er a
re n
ot
stat
isti
call
y
diff
eren
t b
y D
un
can
's m
ult
iple
ran
ge
test
(P
<
0.
05).
At
the
left
let
ters
to
be
read
in
ro
ws/
at t
he
rig
ht
lett
ers
to b
e re
ad i
n c
olu
mn
s.
b F
M-R
MC
-85
bea
n c
ult
ivar
. c
By
mic
ro-k
jeld
ahl.
H
T
=
Har
ves
t ti
me
sam
ple
(m
atu
re d
ry s
eed)
. P
BS
=
P
ho
sph
ate-
bu
ffer
ed s
alin
e.
TC
A
=
Tri
chlo
roac
etic
aci
d.
143
w
A
I - Z LIJ I'- Z 0 0 20
Z
LIJ P 0 n- O. 0 I1"1 F" ~ . / , I1~. ~ - 1
3 7 II 13 Ht
SEED LENGTH (ram)
Fig. 3. Changes in the protein content, expressed as percentage of dry matter, of developing bean seeds, o Total protein content of seed flour by micro-Kjeldahl. [] Protein content of sample extracted by phosphate-buffered saline, precipitated by trichloracetic acid and deter- mined by Lowry.
These disagreements might be ascribed to differences in the chemical basis and interfering substances for each procedure. Micro-Kjeldahl measures protein and nonprotein nitrogen, whereas the Lowry and Bradford methods are intended for protein determination. For Lowry there are two distinct steps which lead to the final color with proteins: (a) reaction with copper in alkali and (b) reduction of the phosphomolybdic-phosphotungstic reagent by the copper-treated protein [8]. There are some interferences for the latter assay system by nonprotein components such as chelating agents, salts, sugars and detergents. For Bradford the binding of a dye to proteins causes a shift in the absorption maximum from 465 to 5~)5 rim, and it is the increase in absorption at 595 nm which is monitored [3]. Compounds such as deter- gents, alkali and guanidine hydrochloride are known to interfere with the color development.
It is interesting to note the dramatic changes undergone by the micro- Kjeldahl nitrogen content during the seed formation, as is shown in detail in Fig. 3. This figure also shows a gradual increase in PBS-soluble/TCA- insoluble protein as determined by Lowry in seeds longer than 7 mm. In
144
Table 2. Protein content a by micro-Kjeldahl and biuret techniques of mature seeds of normal (FM-C-86) and genetically-improved (FM-RMC-86) bean cult±vats
a As percentage of dry matter. In brackets: % recovery from total protein by micro-Kjeldahl. Mean of triplicates + standard deviation. Means with the same letter are not significantly different by Duncan's multiple range test (P < 0.05). PBS = Phosphate-buffered saline. TCA = Tricloroacetic acid.
other words, the PBS-extracted proteins precipitated by T C A increased especially at the last stages of seed development, that is during seed dehy- dration, as assessed by micro-Kjeldahl, Lowry and Bradford methods (Table 1). This behaviour has not been reported previously by other workers and it merits further studies on the type o f proteins synthesised at these stages. For a given procedure, there was a good agreement between the values of PBS-soluble/non-dialysable and PBS-soluble/TCA-insoluble during the seed format ion except for data on 4 mm seeds by micro-Kjeldahl and Lowry methods.
Protein content o f mature bean seeds
To study further the reliability of protein determinat ion methods, the biuret technique was compared to the micro-Kjeldahl procedure with ripened seeds of normal (FM-C-86) and genetically-improved bean materials ( F M - R M C - 86) (Table 2). Fo r bo th cultivars the PBS-soluble/total protein was higher by biuret than by micro-Kjeldahl. By micro-Kjeldahl, the PBS solubilised protein was higher than that extracted in NaCl-ascorbate. Again, according to the biuret method, the PBS-soluble/TCA-insoluble protein was higher than the NaCl-ascorbate soluble/TCA-insoluble sample. All these deter-
Table 3. Protein content a of commercial food proteins
145
Technique Food protein content
Casein Soy protein isolate
Micro-Kjeldahl 92.5 + 0.7 b 84.0 ± 0.2 d Lowry 82.9 + 0.5 d 87.8 + 2.1 c Bradford 93.3 ± 1A b 78.3 ___ 0.7 e Biuret 91.2 + 1.7 b 106.0 + 1.P
As percentage of dry matter. Mean of triplicates + standard deviation. Means with the same letter are not significantly different by Duncan's multiple range test (P ~< 0.05).
minations showed that the FM-RMC-86 material has a higher protein content than the FM-C-86 cultivar.
Protein content of reference food proteins
It was assumed that the main source of the observed discrepancies was the presence of compounds in Phaseolus vulgaris seeds that interfere with the tested analytical procedures; interferences that may be changing in complex- ity during the seed ripening. To test this assumption, protein determinations were performed on reference samples of high protein content; that is highly purified protein samples. Table 3 shows that both agreements and dis- crepancies were also found. Micro-Kjeldahl protein content of casein was 92.5% compared to values of 93.3% by Bradford and 91.2% by Biuret. However, for this same food protein the Lowry procedure gave a value of 82.9%. Surprisingly, when soy protein isolate was tested all the four techni- ques used gave statistically different results. Morr et al. [11] found as well remarkable disagreements in protein content determinations on some food proteins. They found that mean micro-Kjetdahl protein content of soy isolate was 77.9%, whereas by biuret this mean value was 91.2%; and for whey protein concentrate the protein content by the same procedures re- sulted in 35.1 and 70.6%, respectively.
Correlation coefficients
The total protein content of bean flour gave a high correlation coefficient (r = 0.99) with the PBS-homogenate/non-dialysable protein quantitated by micro-Kjeldahl (Table 4). This means that during seed formation the de- crease occurred in total nitrogen was equivalent to the decrease in the non-dialysed nitrogen content. The TCA-precipitated protein estimated by the three determination procedures exhibited very high correlation coef- ficients. For Lowry and Bradford all samples also showed very high corre-
4~
Tabl
e 4.
Cor
rela
tion
coe
ffic
ient
s fo
r pr
otei
n co
nten
t de
term
inat
ion
by m
icro
-Kje
ldah
l, L
owry
and
Bra
dfo
rd t
echn
ique
s
Tech
niqu
e~Sa
mpl
e M
icro
-Kje
ldah
l L
owry
B
radf
ord
PB
S-
PB
S-
PB
S-
PB
S-
PB
S-
PB
S-
hom
ogen
ate/
so
lubl
e/
solu
ble/
so
lubl
e/
solu
ble/
so
lubl
e/
non
tota
l no
n-
TC
A-
tota
l no
n-
dial
isab
le
dial
isab
le
inso
lubl
e di
alis
able
Mic
ro-K
jeld
ahl
Seed
flo
ur p
rote
in/t
otal
P
BS
-hom
ogen
ate/
non-
dial
ysab
le
- P
BS
-sol
uble
/tot
al
PB
S-s
olub
le/n
on-d
ialy
sabl
e P
BS
-sol
uble
/TC
A-i
nsol
uble
Low
ry
PB
S-so
lubl
e/to
tal
PB
S-so
lubl
e/no
n-di
alys
able
PB
S-s
olub
le/T
CA
-ins
olub
le
Bra
dfor
d P
BS
-sol
uble
/tot
al
PB
S-s
olub
le/n
on-d
ialy
sabl
e P
BS
-sol
uble
/TC
A-i
nsol
uble
0.99
-
0.23
-
0.09
-
0.77
-
0.44
-
0.67
-0
.18
-0
.18
-0
.67
-0
.30
-0
.56
-
-0.0
8
0.06
0.
08
-0.1
1
- 0,
70
0.93
0.
78
- 0,
90
0.98
0.92
PB
S-
PB
S-
PB
S-
solu
ble/
so
lubl
e/
solu
ble/
T
CA
- to
tal
non
inso
lubl
e di
alis
able
-0.7
5
-0.6
8
-0.6
6
-0.6
6
-0.5
9
-0.5
7
-0.0
9
-0.1
9
-0.2
0
0.70
0.
74
0.75
0.
99
0.97
0.
97
0,88
0.
88
0.89
0,
99
0.99
0.
99
0.99
0,
99
- 0.
99
PBS-
solu
ble/
TCA
- in
solu
ble
-0.7
3
-0.6
2
0.03
0.
75
0.99
0,93
0,
99
0.99
0.98
0.
97
147
lation coefficients (r >~ 0.88). However, most of these values were statisti- cally different.
Conclusions
The results of this preliminary study suggest that Lowry, Bradford and biuret procedures should not be freely used in protein content determi- nations of bean seeds. Outstanding discrepancies are likely to be found between these techniques and micro-Kjeldahl method; discrepancies that may be found even in isolated food proteins of high purity. On the other hand, the three former methods have advantages over micro-Kjeldahl in their sensitivity and rapidity. These advantages are especially important when a large number of samples need to be screened. Therefore, it was concluded that micro-Kjeldahl method should be used as the reference technique and that Lowry, Bradford or biuret procedures should only be used after they have provided results that agree with data obtained by the reference technique.
Acknowledgement
This research was in part supported by Organizaci6n de los Estados Ameri- canos (OEA)-PRDCYT, and CONACYT-M6xico.
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