THE INOORPORATION OF [35S]OYSTEINE INTO THE PROTEINS OF DOUGH
BY DISULPHIDE-SULPHYDRYL INTEROHANGE
By P. R. STEWART*t and O. M. MAURITZEN*
[Manuscript received September 29, 1965]
Summary
[S6S]Cysteine was added, during mixing, to doughs made from wheat flour,
in amounts which did not significantly affect the level of endogenous diffusible
sulphydryl compounds in flour and which produced no change in the rheological
properties of the dough. The doughs were fractionated by ultracentrifugation
and analysis of the distribution of the isotope in the dough fractions demonstrated
that [S6S]cysteine was bound to both soluble and gluten proteins by disulphide
bonds. [14C]Leucine did not become bound to the fractions of dough under the
same conditions.
Incorporation of cysteine into dough was increased in the a.bsence of air,
suggesting disulphide--sulphydryl exchange rather than oxidation as the mode of
incorporation. The amount of isotope bound to protein was also increased either by
lengthening the times of mixing or relaxation of the dough.
The incorporated [S6S]cysteine could be released in dough only by the addition
of large excess of unlabelled cysteine.
1. INTRODUCTION
Disulphide-sulphydryl interchange has been postulated as the chemical
mechanism underlying the effects of oxidizing, reducing, and sulphydryl-blocking
agents on the rheological properties of dough (Goldstein 1957). Much evidence,
largely of an indirect nature, has been accumulated to support this view (Matsumoto
and ffiynka 1959; Mecham 1959; Frater et al. 1960; Sokol, Mecham, and Pence
1960; Bushuk 1961; Sullivan, Dahle, and Nelson 1961; Bushuk and ffiynka 1962;
Meredith and Bushuk 1962; Mecham, Oole, and Sokol 1963).
Briefly, this theory depicts intermolecular disulphide (-SS-) bonds of gluten
as being critical in the transmission of stress throughout the cohesive protein matrix
of dough. Sulphydryl groups (-SH) , present in much smaller numbers than the
-SS- bonds, may mediate stress release by undergoing interchange reactions (Ryle
and Sanger 1955; Sluyterman 1961) with intermolecula.r -SS- bonds. Hence,
removal of thiol groups (by the use of oxidizing or sulphydryl-blocking agents) or
an increa.se in their number (using reducing agents) should increase and decrease,
respectively, the resistance of dough to applied stress. Reducing agents might also
have a direct effect by decreasing the number of -SS- bonds between protein molecules. Other dough and flour components undoubtedly influence the rheological
properties of dough, but an oxidation-reduction mechanism, involving -SH groups,
* Russell Grimwade School of Biochemistry, University of Melbourne.
t
Present address: Department of Biochemistry, Monash University, Clayton, Vic.
Awt. J. Biol. Sci., 1966, 19. 1125-37
1126
P. R. STEWART AND C. M. MAURITZ EN
appears to be of primary importance in determining the effects brought about by the
agents mentioned above.
The present study attempts to establish directly disulphide-sulphydryl interchange reactions in dough by determining the distribution and mode of binding of
[35S]cysteine in various dough fractions obtained from the preparative ultracentrifugation technique described earlier (Mauritz en and Stewart 1965). The results are
compared with those obtained with [14C]leucine used as an indicator of non-specific
binding in the doughs examined.
The incorporation of [35S]cysteine was also determined under conditions in
which the viscoelastic properties of dough are altered, namely during overmixing
and extended relaxation, and at elevated levels of added thiol. The reversibility
of the incorporation was examined in dough made from one high-quality and one
low-quality flour.
Part of this work has appeared in preliminary form elsewhere (Mauritzen
and Stewart 1963).
II.
EXPERIMENTAL
(a) General Procedure
All reagents used were A.R. grade. The flour used was the commercially
milled Geelong type described previously (Mauritz en and Stewart 1965, 1966).
DL-[35S]Cysteine was prepared from DL-[35S]cystine supplied by Amersham
Radiochemical Centre, England, with an activity of 52 mc/m-mole at the beginning
of these experiments. DL-[l-14C]Leucine (95·6 mc/m-mole) was obtained from the
same source.
Reduction of cystine to cysteine was carried out using, basically, the method of
Benesch and Benesch (1957) and an apparatus of the type described byConsden,
Gordon, and Martin (1947). Recovery of material and extent of reduction were
estimated by measuring polarographically -SS- and -SH groups in the reduced
material (Mauritz en and Stewart 1966). Complete reduction was readily obtained;
recovery varied between 85 and 93%. The labelled DL-[35S]cysteine was stored
in o· 5N H 2SO 4 and an appropriate amount, usually about 0·5 fl-mole with an activity
of 12 fl-c, was neutralized and added to the salt solution, together with carrier where
appropriate, just prior to mixing the dough.
The preparation and fractionation of doughs has been described earlier (Mauritzen and Stewart 1965); the quantity of flour used, however, was decreased to 50 g,
and the volume of O· 5M sodium chloride reduced accordingly to 31· 7 ml. A smaller
mixing bowl was consequently used on the Brabender farinograph.
In the experiments on reversibility of incorporation of [35S]cysteine, the
procedure used was as follows: 50 g of flour was mixed for 3· 5 min with 28·7 ml
of O· 5M sodium chloride containing 0·5 fl-moles (12 fl-c) of [35S]cysteine. The dough
was rested for 10 min and then mixed for a further 3 min with 3·0 ml of o· 5M sodium
chloride containing 500 fl-moles of unlabelled cysteine ("cold chase").
Doughs were mixed in the absence of oxygen, where indicated, by sealing the
mixing bowl of the Brabender farinograph with rubber gaskets and fitting a Perspex
DISULPHIDE-SULPHYDRYL INTERCHANGE IN DOUGH
1127
lid containing gas inlet and outlet vents. Oxygen-free nitrogen was used to flush
oxygen from the flour prior to mixing the dough, and to keep conditions anaerobic
during the mixing process.
Mter preparative ultracentrifugation .of the dough, the dough liquor was
drained off and separated from the contaminating lipid by further centrifugation
(10 min at 8000 g). Layer III, the main gluten fraction was also separated as described
earlier (Mauritzen and Stewart 1965).*
The dough liquor was fractionated further as follows: 4·24 g of dough liquor
was weighed out and diluted with 16·0 ml of 0'5M sodium chloride; as the density
of dough liquor is 1· 06 this represented a 1 in 5 dilution. A 3· O-ml sample of diluted
dough liquor was mixed with 3·0 ml of 10% (w/v) phosphotungstic acid (PTA),
left for 15 min, and filtered through Whatman No. 42 filter paper. A further
4·24 g of dough liquor was weighed directly into a sac made from 18/32 in. Visking
dialysis tubing and subjected to equilibrium dialysis under nitrogen at 2°C for
30 hr against 16·0 ml of O· 5M sodium chloride containing 5 mM ethylenediaminetetraacetic acid.
The original salt solution containing isotope, the diluted dough liquor, the
PTA filtrate, the diffusate, and the non-dialysable material were assayed for radioactivity by plating O' 20-ml samples in duplicate onto disks of filter paper in planchets.
Counting was carried out using a Nuclear Chicago (type D 47) gas-flow counter with
micromil window and type 161A binary scaler. Under these conditions, 1 fLC of
[14C]leucine gave 123,000 counts/min. The time of counting, to not less than 1250
counts (s.d. ±3%), was recorded automatically on a print-out timer.
Preliminary experiments established that the diffusate from dough liquor
could be separated by treatment with PTA into cysteine (soluble in 5% PTA), and
cystine and cystinyl peptides (practically insoluble in 5% PTA).
(b) Calculation of Isotope Distribution
It was not practicable to free the insoluble fractions of dough from contamination with soluble substances (Mauritzen and Stewart 1966); the amount of isotope
bound to insoluble substances in the dough was, therefore, calculated indirectly.
As will be shown later, [14C]leucine did not appear to be bound to the components
of dough and its concentration in dough liquor suggested that it was evenly distributed throughout the total water of the dough. The measured radioactivity of the
added salt solution (31· 7 ml) was therefore corrected for dilution by the endogenous
water content of the flour (6'8 ml).
The difference between this corrected activity and the measured activity of
the diluted dough liquor was expressed as the percentage of added activity bound
to the insoluble components of the dough. The measured activities of the soluble
non-diffusible proteins of dough liquor and of the diffusible cystine (PTA-insoluble)
and cysteine (PTA-soluble) fractions were also expressed as percentages of the
corrected initial isotope concentration.
*The terms "dough liquor" and "layer Ill" refer respectively to the viscous supernatant
and third (major) gluten layer obtained after ultracentrifugation of dough. The preparative
techniques involved are detailed in Mauritzen and Stewart (1965).
1128
P.R. STEWART AND C. M. MAURITZEN
(c) Sulphite Reduction
Samples were reduced by the following procedure: 10-ml portions of dough
liquor or 2-3 g of layer III from radioactive doughs were dispersed in 25 ml of 8M
urea. These samples were then dialysed against changes of 8M urea until no further
activity appeared in the diffusate. This treatment removed the diffusible material
of dough liquor or contaminating diffusible material in layer III. The dialysis sacs
were then opened and samples removed for plating and counting. 10-ml portions
of the contents of the sac were dialysed against 8M urea containing O· 25M sodium
sulphite. Samples were also dialysed against 8M urea containing o· 5M sodium chloride
in order to detect any loss of label due to ionic displacement. After 48 hr and five
changes (40 vol. per change) at 2°C, the sacs were opened, sampled, and counted.
Correction was made for self-absorption in samples containing sodium sulphite.
(d) Performic Acid Oxidation
Oxidative cleavage of disulphide bonds was performed using the method of
Hirs (1956), except that the concentration of formic acid was decreased by 30%
(dilution by sample) and the reaction time doubled to 60 min. Hirs detected no peptide
hydrolysis under the conditions used by him.
Dough liquor (7 g) from radioactive dough was mixed with 14 ml of 18N formic
acid and dialysed against 13N formic acid until no further activity appeared in the
diffusate. 3 g of layer III from radioactive dough was dispersed in 25 ml of o· 02N
formic acid and dialysed in the same way. The dialysis sacs were then opened and
samples plated for counting. lO-ml portions from the sacs were treated with 0·45 ml
of 39% hydrogen peroxide and left at room temperature for 60 min. Dialysis was
then recommenced against 13N formic acid and continued for 48 hr with five changes
(20 vol. per change) at 2°C. Control samples (10 ml), untrea.ted with hydrogen peroxide,
were similarly dialysed. The contents of the bags were then sampled, plated, and
counted.
III.
RESULTS AND DISCUSSION
When 1 ·0 /Lmole of [14C]leucine (5·6 /Lc) was added to dough, it was recovered in
dough liquor in amounts almost equal (96-99%) to those which would be expected
if no binding of this amino acid to the insoluble components of dough occurred, and
if the whole of the water content of the dough [i.e. water added plus water present
in the flour (13'6% by weight)] was available for its solution.
It is possible that the endogenous water of the flour was not accessible to
leucine, that binding of leucine to the insoluble substances of dough occurred, and
that this binding of leucine exactly balanced the presumed dilution of the isotope
by the endogenous water. However, the concentration of added [14C]leucine in
dough liquor was exactly the same as in a control in which the doughs were swamped
during mixing with unlabelled leucine or cysteine. The latter, at the level of 500
/Lmolesj50 g flour, caused a drastic alteration to the viscoelastic properties of the
dough. These structural alterations to the dough matrix would ha.ve been expected
to have exposed additional binding sites for the absorption of [14C]leucine. Moreover,
1129
DISULPHIDE-SULPHYDRYL INTERCHANGE IN DOUGH
dialysis against 8M urea of either dough liquor or the gluten layer III from a fractioned,
[14C]leucine-treated dough resulted in the loss, by dialysis, of all but 1-2% of the
initial radioactivity of the fraction.
Interaction of leucine with dough components would most probably occur
through its amino and carboxyl groups, rather than its weakly hydrophobic sidechain. The small amount of binding of leucine observed might account for a small
part of the binding of cysteine added to dough.
(a) Mode of Binding of [35S]Cysteine in Dough
When 0·5 fLmole of [35S]cysteine was added to dough and the dough subsequently fractionated, the recovery of label in dough liquor was 20-40% lower,
depending on the mixing and resting conditions used, than that in the case of
[14C]leucine.
Typical values for the distribution of [14C]leucine and [35S]cysteine in fractions
of doughs which had been mixed for 3·5 min in air and rested for 60 min prior to
fractionation are given in Table 1.
TABLE I
DISTRIBUTION OF [I<C]LEUCINE AND [35S]CYSTEINE IN VARIOUS DOUGH FRACTIONS
Percentage of Added Isotope in Dough Fraction
Isotope Added
Gluten
Proteins
[14C]Leucine
[35S]Cysteine; mixed
in air
[35S]Cysteine; mixed
under nitrogen
I
Soluble
Proteins
Diffusible/
PTA-insoluble
Fraction
Diffusible/
PTA-soluble
Fraction
4
0
10
86
24
11
30
35
29
21
7
43
The term "gluten protein" is used in a general sense and signifies all the protein
nitrogen of dough not accounted for by dough liquor. "Soluble protein" denotes the
non-diffusible fraction of dough liquor and includes the carbohydrates of this fraction
since no data were obtained on the relative radioactivity of the protein and carbohydrate fractions of dough liquor. A number of workers have noted the powerful
association between the soluble proteins and carbohydrates of flour (Pence, Elder,
and Mecham 1950; Udy 1953; Waldschmidt-Leitz and Hochstrasser 1961). It is
doubtful, however, that carbohydrates as such would form covalent linkages with
cysteine under the conditions used. The "diffusible/PTA-insoluble" fraction represents cystine or cystinyl peptide material in dough, while values for unreacted
cysteine, and possibly small quantities of smaller cystinyl peptides, are given by
the "diffusible/PTA-soluble" fraction.
There are two possible major binding sites of [35S]cysteine to the insoluble
fraction of dough, namely starch and gluten. The former was eliminated when it was
1130
P. R. STEWART AND C. M. MAURITZEN
found that the isotope could be completely removed along with gluten by extraction
of layer III (starch plus gluten) with 0·02N formic acid and sedimentation of the
insoluble starch.
Thus, both soluble and insoluble proteins of dough bind [35S]cysteine. The major
possible reactions by which this binding could occur are:
1.
Protein-SS-Protein +Cy-SH~Protein-SS--cy + Protein-SH.
•
••
2(a). 2 Cy-SH+[O]--+Cy-SS-Cy+H20 .
•*
*
*
2(b). Cy-SS-Cy + Protein-SH~Protein-SS-Cy +Cy-SH.
•
•
3.
Protein-SH+Cy-SH+[O]--+Protein-SS-Cy+HzO.
Reactions 1 and 2(b) represent two forms of -SS-/-SH interchange. Reaction 1 is
the mechanism proposed to account for stress release in the insoluble protein matrix
of dough. Reactions 2(a) and 3 are oxidations to which most sulphydryl compounds
are susceptible.
TABLE 2
RELEASE OF ISOTOPE FROM THE SOLUBLE AND GLUTEN PROTEINS OF DOUGHS TREATED WITH
[1'C]LEUCINE AND [36S]CYSTEINE
Dough Fraction
Layer III proteins
Layer III proteins
Layer III proteins
Soluble proteins
Soluble proteins
Soluble proteins
Layer III proteins
Layer III proteins
Soluble proteins
Soluble proteins
Layer III proteins
Soluble proteins
Isotope
Added
Dialysis Medium
Original Activity
Remaining
(%)*
[l4Q]Leucine
[35S]Cysteine
[85S ]Cysteine
[1'C]Leucine
[86S]Cysteine
[86S]Cysteine
[36S]Cysteine
[36S]Cysteine
[36S]Cysteine
[36S]Cysteine
[36S]Cysteine
[36S]Cysteine
8M Urea
8M Urea
13M Formic acid
8M Urea
8M Urea
9M Formic acid
O' 5M Sodium chloride in 8M urea
o. 25M Sodium sulphite in 8M urea
o.5M Sodium chloride in 8M urea
o.25M Sodium sulphite in 8M urea
13M Formic acid+H.O s
9M Formic acid+HsOs
1·2
97
91
1·2
100
100
99
24
95
24
36
27
* After exhaustive dialysis.
Whichever of these mechanisms occurs in dough, [35S]cysteine would become
bound to protein by disulphide bonds. The release of label should then only be
possible in the presence of reactants which cleave disulphides: e.g. sulphite and
performic acid.
The results obtained are summarized in Table 2. It is evident that the label
was bound by -SS- bonds both to the soluble protein and the layer III proteins,
since about 70% of the bound activity became diffusible on treatment with sulphite
or with performic acid. Very little was released, on the other hand, in the presence
of high concentrations of urea, formic acid, or sodium chloride in.urea, treatments
DISULPHIDE-SULPHYDRYL INTERCHANGE IN DOUGH
1131
which disrupt ionic or hydrogen bonds. This suggests that little or no binding of
[35S]cysteine occurs through such bonds.
In the same table, results from the dialysis of dough liquor and samples of
layer III from [14C]leucine-treated doughs are presented. Dialysis against O' 5M
sodium chloride or 0'02N formic acid (dough liquor and layer III, respectively) gave
practically identical results in that 98-100% of the activity was diffusible, and it
is therefore quite apparent that leucine was only loosely associated, if at all, with
the non-diffusible components of dough.
(b) Mechanism of the Incorporation of [35S]Cysteine into Proteins of Dough
The three reaction mechanisms (reactions 1, 2, and 3 above) by which it is
proposed [35S]cysteine may be incorporated into the proteins of dough have important
differences. Only reaction 1, involving two protein molecules, can be expected to
influence directly the viscoelastic properties of dough, since the transmission of
stresses occurs through covalent bonds between the insoluble gluten proteins.
Peptides and amino acids, because of their size, are unlikely to be involved, structurally, in a three-dimensional protein network, and hence are not directly concerned
in stress transmission in dough. Reactions 2 and 3, which involve only single protein
chains, are therefore unimportant in this respect. Rea.ctions 2(a) and 3 depend on
the presence of an oxidant. In dough mixed in air, this role is played ultimately by
oxygen. Smith and Andrews (1957) have demonstrated the considerable uptake of
oxygen by doughs during mixing. Aerial oxidation was restricted by mixing dough
under nitrogen, but this, of course, would not prevent the action of other oxidants
in dough, such as heavy metal ions, peroxides etc. Nor could it prevent the formation
of cysteine by disulphide-sulphydryl interchange thus:
4.
Protein-SS-Cy +Cy-ElH ~Protein-SH +Cy-SS-Cy.
When an otherwise normal dough from Geelong flour was mixed under nitrogen,
the distribution of added [35S]cysteine was considerably different from that of a
dough mixed in air, as can be seen from the values in Table 1. A greater proportion
of the total added radioactivity was found in both soluble and gluten protein fractions,
particularly the former where incorporation was almost doubled in the case of the
dough mixed under nitrogen compared with the control mixed in air.
It is also evident from the values in Table 1 that the increased anaerobic
binding of [35S]cysteine to proteins has been largely at the expense of the cystine
fraction (diffusible, PTA-insoluble) which has fallen from 30 to 7%, and not of the
unchanged· cysteine which has increased from 34 to 43 % of 'the initial activity.
These results support the following conclusions:
(1) The mode of incorporation of [35S]cysteine into protein of dough is predominantly by disulphide-sulphydryl interchange involving protein
disulphide bonds.
If incorporation occurred via oxidation to cystine and exchange with
protein sulphydryls, or by oxidation of [35S]cysteine with protein sulphydryls
[reactions 2(a), 2(b), and 3 above], then the absence of oxygen should
decrease the incorporation of isotope into protein rather than increase
it, as was found to be the case.
1132
P. R. STEWART AND C. M. MAURITZEN
(2) Extensive oxidation of cysteine to cystine occurs in air-mixed doughs
since only 7% of the added isotope was located in the diffusible, PTAinsoluble fraction when dough was mixed under nitrogen compared with
30% when mixed in air.
(3) While both cysteine and cystine may be incorporated into proteins by
reactions 1 and 2, respectively, it would appear that cysteine is more readily
incorporated; this in turn may be because of the overall preponderance of
protein disulphide over sulphydryl groups in dough.
(4) Mixing under nitrogen, which decreased the oxidation of cysteine to cystine,
also resulted in approximately double the incorporation of [35S]cysteine into
the soluble protein fraction, while under the same conditions the incorporation into gluten was increased only by approximately 20%. This would
suggest that soluble proteins are more susceptible than gluten to disulphidesulphydryl interchange under normal conditions.
In the experiment described above, the amount of [35S]cysteine added, 0·5
p.mole, to 50 g of flour is unlikely to alter significantly the endogenous levels of
diffusible sulphydryl groups since this amount of flour contains at least 6-7 p.moles
of such -SH groups (Mauritzen and Stewart 1966). Moreover, no rheological changes
were observed at this level of added [35S]cysteine.
The ratio of flour -SS- bonds to endogenous diffusible -SR groups, including
the added [35S]cysteine, is about 120 to 1, but from the distribution oflabel in normal
dough noted above, more than one-third of the added [35S]cysteine remained unreacted. This confirms the earlier results (Mauritzen and Stewart 1966) that there
are significant quantities of diffusible sulphydryl material, originally present in
flour, which survive oxidation and -SS-/-SH interchange during the mixing process
and may be recovered in dough liquor.
Since it has been postulated (Frater et al. 1960) that the amount and reactivity
of -SH groups in dough influence its viscoelastic behaviour and that these groups
exert their effect through -SS-/-SH interchange reactions, it seemed appropriate
to investigate the incorporation of [35S]cysteine into dough proteins under conditions
which might alter the rate or extent of such interchange reactions, namely increased
mixing and resting times.
(c) Mixing Time
If disulphide-sulphydryl interchange is the mechanism of stress release which
operates during mixing, then increased time of mixing (at constant resting time)
should increase the incorporation of [35S]cysteine into the gluten proteins of dough.
Both increased time of reaction (assuming the reaction is not at equilibrium), and
increased exposure of unreacted disulphide bonds by the action of mixing could
account for this.
The alterations in distribution of label which occurred when otherwise normal
doughs were mixed for 30 min and 60 min are shown in Table 3.
Incorporation into the gluten proteins occurred linearly with increasing mixing
times at the expense of all three fractions of the dough liquor. This may indicate
that the proteins of dough liquor, as well as diffusible components, are bound to the
1133
DISULPHIDE-SULPHYDRYL INTERCHANGE IN DOUGH
gluten proteins during mIxmg. The soluble proteins, while probably of no direct
importance to the physical structure of dough, may thus have an important role in
modifying the viscoelastic properties of the insoluble gluten proteins.
TABLE
3
EFFECT OF INCREASED MIXING TIMES ON THE DISTRIBUTION OF ADDED [35S]CYSTEINE
IN FRACTIONS OF DOUGH MADE FROM GEELONG FLOUR
Percentage of Added Isotope in Dough Fractions
Mixing Time
(min)
Gluten
Proteins
Soluble
Proteins
Diffusible/
PTA-insoluble
Fraction
Diffusible/
PTA-soluble
Fraction
24
11
30
:15
30
32
7
30
:n
60
38
4
28
30
3·5
(d) Resting Time
If disulphide-sulphydryl interchange is responsible for relaxation of stress in
doughs during resting, then provided the interchange reaction of [35S]cysteine with
protein disulphide bonds in the dough is not at equilibrium, incorporation of label
into the gluten proteins should increase with increased resting time.
o
o
w
o
o
«
"- 20
-----.
o
w
""
I-
~
10 1'----'----'
U
0:
W
lL
o
2
4
6
8
10
12
14
16
RESTING TIME (HR)
Fig. I.-Effect of increased resting time on the incorporation of
added [35S]cysteine into fractions of dough at constant mixing time
of 3·5 min. X Gluten proteins. 0 "Diffusible/PTA-soluble" fraction .
.... "Diffusible/PTA-insoluble" fraction. • Soluble proteins.
Otherwise-normal doughs were subjected to increasing resting times of up to
15 hr. The effect of this treatment on the distribution oflabel is illustrated in Figure l.
Incorporation into the gluten proteins increased with increasing relaxation time and
followed much the same pattern as the relaxation of strains in dough-rapid initially,
1134
P. R. STEWART AND C. M. MAURITZEN
then much slower. However, compared with the 45-60 min required for almost
complete stress relaxation in dough, as measured by the extensograph (Frater et al.
1960) incorporation of label proceeded for 3-4 hr before falling off. This suggests
that it may only be in the early period of -SS-j-SH interchange that disulphide
bonds transmitting stress are broken. These may be the intermolecular disulphide
bonds of gluten. In the later stages of incorporation of [35S] cysteine , bonds not
important in stress transmission throughout the dough, for instance protein-peptide
and possibly intramolecular protein disulphide bonds, may be more involved in
interchange reactions.
1400
E1200
o>:
Q1000
>u
E
~
800
a
3
s:
~
0
600
u
(;
400
~
0
"""
200
AMOUNT OF UNLABELLED CYSTEINE ADDED (!(MOLES)
Fig. 2.-Effect of increasing concentrations of unlabelled cysteine
on the incorporation of added [35S]cysteine into various fractions
of dough. • "Diffusible/PTA-soluble" fraction. 0 "Diffusible/
PTA-insoluble" fraction . .A Gluten proteins. X Soluble proteins.
Incorporation occurred almost completely at the expense of the two diffusible
fractions, the soluble proteins apparently being little involved in the relaxation
process.
(e) Level of Sulphydryl Groups in Dough
Other studies (Frater et al. 1960; Frater, Hird, and Moss 1961) have detailed
the rheological alterations which occur when the level of sulphydryl groups in dough
is increased.
The incorporation of [35S]cysteine into the proteins of dough at levels of added
cysteine which gave a rheological response was examined by adding increasing
amounts (100-1500 p,moles) of carrier cysteine, with a constant amount of [35S]cysteine
(0·5 p,mole, about 12 p,c) to otherwise normal doughs. This permitted direct comparison of the radioactivity of dough fractions at different levels of added cysteine.
Doughs made from the Geelong flour used in these experiments gave a detectable
1135
DISULPHIDE-SULPHYDRYL INTERCHANGE IN DOUGH
response (lower strength, greater extensibility) at a level of added cysteine of
15 f-LmolesJ50 g of flour.
From Figure 2 it can be seen that the gluten proteins incorporated cysteine
almost linearly until 1000 f-Lmoles of cysteine per 50 g of flour had been added. At
this point 200 f-Lmoles of the added cysteine were present in the gluten fraction, but
further increase in the level of cysteine added to the flour resulted in no further incorporation into this fraction; in fact a slight decrease of bound [358]cysteine was
observed. Hence, of the 330 f-Lmoles of -88- bonds present in the gluten proteins of
this dough, about 60% were reactive towards added cysteine under the conditions
used. Reaction of the residual-88- bonds may, however, come about with increased
mixing time, as a result of exposure of further gluten molecules, or disruption of
protein structure.
TABLE
4
DISTRIBUTION OF ADDED [35S]CYSTEINE IN VARIOUS FRACTIONS OF DOUGH COMPARED WITH SIMILAR
FRACTIONS FROM DOUGH TO WHICH AN EXCESS OF UNLABELLED CYSTEINE HAD BEEN ADDED
Percentage of Added Isotope in Dough Fractions
Flour
Dough
Treatment
Gluten
Proteins
Soluble
Proteins
Diffusiblej
PTA· insoluble
Fraction
Diffusible/
PTA· soluble
Fraction
Geelong
Normal
26
10
30
34
Geelong
"Cold chase"
15
1
7
77
Gabo
Normal
31
9
28
32
Gabo
"Cold chase"
23
3
12
62
-----"-
-------
Examination of the incorporation of [358]cysteine into dough fractions over
a narrower range of added cysteine (0·5-100 f-Lmoles) than that shown above (0·5-1500
f-Lmoles) was made in an effort to detect differences in the reactivity of the -88bonds of the dough proteins. Incorporation into the gluten proteins and the diffusible
fractions was approximately linear over the range of added cysteine. The soluble
proteins on the other hand behaved quite differently. Cysteine incorporation
increased until 15 f-Lmoles per 50 g of flour had been added to the dough; after this
there was no further incorporation into the soluble proteins, and the results show
that only about 1 f-Lmole [358]cysteine reacted with 140 f-Lmoles of -88- bonds present
in this fraction. The reactivity of the --88- bonds of the soluble proteins is therefore
quite different from that of the gluten proteins.
The resistance of the disulphide bond of the soluble proteins to reaction with
[ 358]cysteine parallels the observations of Hird and Yates (1961) who found that
only 50% of the disulphide bonds of solutions of the water-soluble proteins of flour
1136
P. R. STEWART AND C. M. MAURITZEN
were reduced by sodium borohydride, whereas all of those of the gluten proteins
were susceptible to treatment with this reagent.
(f) Reversibility of Incorporation
The distribution of [35S]cysteine in doughs made from Geelong and Gabo flours
and subsequently swamped with unlabelled cysteine ("cold chase") is shown in
Table 4. A comparison with the values for normally treated doughs shows that
there was a considerable reduction in the amount of bound isotope in both protein
fractions; the gluten fraction, in the case of the Geelong dough, had lost 40% of its
activity and the soluble protein approximately 90%. While this demonstrates the
essential reversibility of the reaction it should be remembered that a thousand-fold
excess of "cold" cysteine was used. A 20-fold excess of unlabelled cysteine under
the same conditions produced no detectable loss of isotope from the protein fractions,
while a 100-fold excess of cysteine caused only a slight loss of activity from the
soluble protein.
(g) Varietal Differences
A single varietal comparison of the incorporation of [35S]cysteine into the
proteins of dough was made between normal doughs from Geelong and Gabo flours,
two flours differing widely in rheological characteristics (Mauritzen and Stewart
1965). The pattern of incorporation observed is shown in Table 4; no gross quantitative differences were found. The presence of unreacted cysteine in dough from Gabo
flour is interesting, since it was found earlier (Mauritz en and Stewart 1966) that no
free diffusible sulphydryl groups could be detected in such doughs. This suggests
that, in contrast to Geelong, Gabo flour contains no, or very little, diffusible sulphydryl
material.
IV.
ACKNOWLEDGMENT
The authors wish to thank the Wheat Industry Research Council for a grant
from which the expenses of this investigation were defrayed.
V.
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