Journal of Cereal Science 14 (1991) 243-256

Changes in Electrophoretic Patterns of Acetic Acid­insoluble Wheat Flour Proteins During Dough Mixing

D. SIEVERT, H. D. SAPIRSTEIN and W. BUSHUK

University of Manitoba, Food Science Department, Winnipeg, Manitoba,Canada R3T 2N2

Received 4 June 1990

Acetic acid-insoluble (residue) protein fractions ofwheat flours and respective doughswere studied by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS­PAGE). Gel patterns of unreduced SOS buffer extracts from residue fractions showedthat dough mixing caused systematic changes in the staining intensity of a group ofrelatively high molecular weight proteins comprising triplet band proteins andneighboring bands. Together, these proteins were named in this study the triplet zoneproteins. Continued mixing of control doughs resulted in a progressive decrease in theintensity of the triplet zone bands, whereas mixing in the presence ofpotassium iodate(KIOa) and N-ethylmaleimide (NEMI) caused an increase in staining intensity ofthese bands. These results appear to be related to sulfhydryl-disulfide interchangereactions that have been proposed to occur during dough mixing. Results for doughsfrom water-extracted flour suggested water soluble components of the flour interactedduring mixing with the triplet zone proteins. SDS-PAGE patterns of reduced proteinsthat could not enter the separating gel under non-reducing conditions (slot protein)resembled the patterns of purified glutenin. Electrophoretic patterns of slot protein of80S buffer extracts from acetic acid-insoluble residues showed the presence ofsubunits that corresponded in molecular weight to subunits of triplet band proteins.This suggested the possibility of strong interaction between triplet band proteins andSDS·buffer extractable glutenin in flour and dough residues.

Tntroduction

Previous work has established a relationship between the amount of residue protein thatremains after flour extraction with non-reducing solvents and mixing strength of wheatflour doughsl-7. The residue fraction is affected by dough mixing; mixing was shown toinduce a characteristic decrease in the proportion of urea-insoluble proteinS or diluteacetic acid-insoluble protein2,3,6,7,9 and an analogous increase in the proportion ofsoluble protein. Size exclusion chromatography results indicated that the increase in theamount of soluble protein during dough mixing could be accounted for by an increasein the amount of extractable glulenin6, 7,10,11. Electrophoretic studies showed nosignificant effects of mixing on SDS-PAGE patterns of reduced Osborne fractions orreduced acetic acid-soluble and -insoluble glutenin proteins6

. Differences, however, weredetected in the lower molecular weight region (Mr 16-26k) of unreduced acetic acid­soluble protein fractions of under- and optimally-mixed doughs9

•

Publication no. 200 of the Food Science Department, University of Manitoba.

0733-5210/91/060243+ 14 $03.00/0 © 1991 Academic Press Limited

244 D. SIEVERT ET AL.

We recently characterized by one- and two- dimensional electrophoresis, SDS buffer­extractable protein from acetic acid-insoluble residues of wheat flour12 • The object ofthis study was to examine further the effects of mixing on the electrophoretic pattern ofacetic acid-insoluble proteins of doughs from wheat varieties with diverse mixingproperties.

ExperimentalMaterials

Wheat cultivars used in this study included four hard red spring (Neepawa, Katepwa, Glenlea,Pembina), two soft white spring (Kenya, Fielder), and one soft white winter (Augusta). The grainwas milled into straight grade flour on a Buhler laboratory mill. Flour protein contents(N x 5·7, 14% mb) were: Neepawa, 14·9 %; Katepwa, 13·7 %; Glenlea, 12·9 %; Pembina, 13·4 %;Kenya, 12·0%; Fielder, 8·8%; and Augusta, 8·0%.

Preparation of water extracted-flour

For water extraction of flour (Neepawa only), we used a I: 10 (wIv) flour :water ratio as describedby Shuey and Gilles13

• The flour (50 g) was suspended in 500 ml distilled water, stirred on amagnetic stirrer for 2 hat 4 DC and then centrifuged for 30 min at 10000 g (4 DC). The supernatantwas decanted and the water-insoluble residue was freeze-dried and subsequently ground using amortar and pestle.

Preparation of dough samples

Doughs were mixed in a Brabender Farinograph in air using 50 g (14% mb) of flour. Doughs wereprepared from flour with no additives (control) and with addition (in total water) of 30 ppmpotassium iodate (KIOa) and 60 ppm N-ethylmaleimide (NEMI). Dough mixing was also carriedout with the water-extracted flour of the Neepawa cultivar. For each dough, duplicate sampleswere mixed for I min (premix), peak dough development time, 15 min and 30 min. Doughs werefrozen in liquid nitrogen, freeze-dried and finely ground using a mortar and pestle.

Extraction and fractionation ofproteins

Acetic acid-insoluble residues of the flours and freeze-dried doughs were prepared by extractingI g sample (14 % rnb) with 0·05 M acetic acid (25 mI) as described by Orth and O'Brien5• Theresidues were freeze-dried, weighed and analysed for moisture and protein (N x 5·7).

For SDS-PAGE analysis, flour and residue samples containing 6 mg protein (N x 5·7) wereextracted with 1 ml of2 % (wjv) SDS buffer solution as described by Ng and Bushuk14 • Extractionwas overnight (16 h) at room temperature in 15 ml centrifuge tubes, with occasional shaking ona vortex mixer, and subsequent centrifugation at 20000 g and 20 DC for 20 min. The supernatantwas decanted into a microcentrifuge tube, heated at 100°C for 3 min, and clarified by centrifuging(10 min, 12000 g, room temperature). SDS buffer extracts of acetic acid-insoluble residues wereprepared without and with 5 % (vjv) of the reducing agent 2-mercaptoethanol. SDS bufferextraction of the residues without mercaptoethanol was followed by an SDS buffer extraction withmercaptoethanol. Aliquots of each extract were analysed by SDS-PAGE.

SDS-PAGE

SDS-PAGE was carried out on an LKB 2001 unit (Pharmacia LKB) according to a modifiedLaemmli procedure14 • The stacking and separating gel concentrations were }03 and 17·3 %,respectively. Gel thickness was 1·5 mm. Unless stated otherwise, electrophoresis was carried outfor 45 min at 35 rnA and then at 10 rnA until the tracking dye reached the bottom of the gel

RESIDUE PROTEIN AND DOUGH MIXING 245

('normal' conditions). To improve resolution of low mobility proteins in some experiments,electrophoresis was carried out for 45 min at 35 rnA, 18 h at 20 rnA and 1 h at 30 rnA (' extended'conditions).

A two-step one-dimensional SDS-PAGE procedure was performed as described by Singh andShepherd15. For the first step, an unreduced protein extract was loaded onto a 1·0 mm thick gel,and electrophoresed. The top section of the separating gel (0'5 cm from the origin), whichcontained unresolved proteins, was cut out and equilibrated in a solution containing 2 % (vIv) 2­mercaptoethanol for 1·5 h at 37 °C16, and subsequently loaded onto a thicker (1'5 mm) gel for thesecond electrophoretic separation. Electrophoresis of the reduced proteins was carried out at35 rnA for 45 min, and at 10 rnA until the tracking dye migrated to the end of the gel.

Apparent molecular weights were estimated using the following reference proteins (SigmaChemical Co., 8t Louis, Missouri): myosin (205k), ~-galactosidase (I 16k), phosphorylase b(97'4k), bovine serum albumin (66k), egg albumin (45k), carbonic anhydrase (29k), trypsininhibitor (20'lk) and lysozyme (l4·3k). A reduced protein extract of Neepawa flour was used asa secondary standard 17• Electrophoretic patterns of reduced protein extracts were divided into fiveregions (RI-R5) that correspond to classes of endosperm proteins as proposed by Fullingtonet a/. 1S : R 1, high-molecular-weight (HMW) glutenin subunits; R2, almost entirely co-gliadins; R3,mainly LMW glutenin subunits, but with some CI.-, po, y-gliadins; R4, mainly CI.-, po, y-gliadins, butwith some LMW glutenin subunits; and R5, albumins with small amounts of globulins.

Results and Discussion

Comprehensive results for one wheat variety, Neepawa, representing the CanadaWestern Red Spring class, are presented. Selected comparative results for other wheatvarieties are reported where appropriate.

SDS-PAGE of extracts prepared under non-reducing conditions

Control doughs. Electrophoretic patterns of unreduced SDS-buffer extracts of flour anddough samples (cv. Neepawa), taken at different stages of mixing, provided someindication that a group of relatively high molecular weight (HMW) proteins known as'triplet band' proteins (triticin)15.19, was affected by dough mixing. With increasingmixing time, we observed a slight decrease in the staining intensity of these bands (resultsnot shown). Differences in the electrophoretic patterns were confined to the tripletbands; other bands were not affected.

To determine whether residue protein showed any SDS-PAGE pattern variationduring dough mixing, we prepared SDS buffer extracts (without mercaptoethanol) of0·05 M acetic acid-insoluble residues of flour and doughs. As expected from previouslypublished information6• 7, the protein content of the residue fractions decreased withincreasing mixing time (Table I). This decrease was taken into account in theelectrophoretic analyses of the samples by adjusting the quantity of the freeze-driedresidue that was extracted with SDS buffer to contain a constant amount of protein.

Compared with the patterns of the extracts of flour and doughs, the patterns of theresidue fractions [Fig. leA)] showed a greater decline in band staining intensity of thetriplet band proteins during mixing than did the patterns of extracts of the freeze-drieddoughs. Triplet bands, as well as the level of background streaking, were most intensein the tracks of the extract of the 1min mixed dough [Fig. l(A), lane (b)]. As mixingcontinued, these bands gradually became fainter. The reduction in staining intensity wasespecially noticeable after 15 and 30 min of mixing. A similar effect was observed for a

246 D. SIEVERT ET AL.

TABLE 1. Yield and protein content of acetic acid-insoluble residue fractions from Neepawaflour and doughs mixed in the absence (control) and presence of K103'

Dough mixing time (min)

Flour 5 IS 30

Yield (g)Control 0'83 0·86 0·84 0·84 0·83KI03 0·85 0'89 0·85 0·84 0·84

Protein content (%)Control 6·1 5'7 4·8 4-4 4'1KI03 6·0 6·6 4·2 3·1 2·8

Proportion (%) of total flour proteinControl 29·2 28·3 23-3 21·3 19-6KI03 29·4 33'9 20·6 15·0 13-6

Values are on a dry matter basis, with 1·0 g starting material. Based on duplicate determinations, meanvariation in residue yield and protein content was ±O·02 g and ±O'2 %, respectively.

~

Tripletbonds [

:::=J

•

Q -·f3-,y-r<;)lIodins

abc d e a b c' d e(Al (B) (e)

FIGURE 1. SDS-PAGE of SDS buffer extracts prepared under non-reducingconditions from acetic acid-insoluble residues of control (A) and iodate-treateddoughs (B). Lanes correspond to (a) Neepawa flour, and doughs mixed for (b) I min,(c) 5·5 min, (c') 4 min, (d) 15 min and (e) 30 min. Lane (C) is a photographicenlargement of the pattern of an SDS buffer extract that corresponds to lane (e) in

(B). Electrophoresis was performed under extended conditions.

RESIDUE PROTEIN AND DOUGH MIXING 247

neighbouring band with lower mobility [Fig. 1, open-headed arrow] and for two faintbands with slightly higher mobility [Fig. 1, solid arrows] which together with the tripletband proteins will be here referred to as 'triplet zone' proteins. A more distant band withmuch higher mobility [Fig. 1, triangle] showed a decrease in intensity similar to thatobserved for the triplet zone proteins. In contrast, an opposite pattern effect of mixingwas observed for the a-, P-, y-gliadin bands, which increased in staining intensity withmixing following a slight decrease after 1 min of mixing. Leaving a 1 min-mixed doughfor 30 min at 30°C or incubating a flour suspension (1: 10 w/v flour:water ratio) at30°C for 30 min did not cause any changes in the electrophoretic patterns of the extractsof the acetic acid-insoluble residues. Thus, mixing is required to induce the events shownin Fig. I.

To obtain information on possible intercultivar variations, the residue fractions ofseven other common wheat cultivars were analysed as reported above for Neepawa.Irrespective of the cultivar used, we observed a systematic decrease in intensity of tripletzone proteins with mixing; this effect was slightly more pronounced for cultivars withlow mixing tolerance (e.g. Fielder, Augusta, Pitic). A less consistent effect amongcultivars was observed with regard to the a-, P-, y-gliadin bands. Residue fractions ofthecultivars Katepawa, Fielder and Pitic, like Neepawa, showed a definite increase ingliadin band staining intensity with mixing. On the other hand, Glenlea, Pembina,Kenya and Augusta showed only minor changes in the intensity of gliadin bands.

It should be noted here that the efficiency of extraction of residue protein withoutreduction does not appear to be a factor in any of the above noted effects. Repeatedextraction of a flour or dough residue with SDS buffer showed the presence of tripletzone proteins and a-, p-, y-gliadin bands in the first and second extracts only (results notshown). The question therefore arises as to what might be the reason for the consistentlyobserved changes during mixing of the triplet zone proteins. Dough mixing is generallyviewed as a process that involves disaggregation and/or depolymerization of aggregatedor polymeric proteins, thereby increasing their extractability. SDS-PAGE patterns ofunreduced acetic acid-soluble protein extracts of flour and doughs showed no tripletzone bands (results not shown). Hence, the mixing-related decrease in intensity of thetriplet zone bands in patterns of extracts of acetic acid-insoluble residues was not due toan increase in their extractability from the doughs with acetic acid. Accordingly, theresults suggested that either depolymerization and/or association of triplet zone proteinswith other dough constituents were responsible for the observed decrease in bandintensity of these proteins.

Doughs mixed in the presence of KlO a and NEMI. Analogous acetic acid extraction offreeze-dried doughs and SDS-PAGE analyses of the residues were carried out fordoughs mixed in the presence of the oxidizing improver potassium iodate (KIOa). Uponextraction of iodate-treated doughs with acetic acid, less protein remained in the residuethan in the residues of the analogous control doughs (see Table I). This is in generalagreement with previous reports6- 8 , which showed a considerable increase in theproportion of extractable protein from doughs mixed in the presence of iodate.

In striking contrast to the electrophoretic patterns of SDS buffer extracts of acetic acidresidues from control doughs [Fig. leA)], corresponding patterns from iodate-treated

248 D. SIEVERT ET AL.

doughs indicated an opposite trend in the triplet band region [Fig. I(B)]. With increasingmixing time, an increase in the staining intensity of triplet zone proteins and of the bandwith higher mobility (Fig. 1, triangle) was observed. The pattern of the SDS bufferextract of the dough residue after 30 min mixing with iodate [Fig. l(B), lane (e)] alsoindicated the appearance of additional bands that were not present or were very faint inthe patterns of the flour residue and the residues of doughs mixed for a shorter time.Figure I(C) (arrow-heads) shows this result more clearly. Except for the triplet zonebands, no other differences were observed between gel patterns of control and iodate­treated doughs. Both sets of patterns showed an increase in ('j,-, ~- and y-gliadin bandsas mixing proceeded.

Triplet zone proteins extracted from residues of the other wheat cultivars examinedgave a similar response to mixing in the presence of iodate as did Neepawa. As mixingcontinued, bands in the triplet region gradually became more intense.

When iodate was added to the dough in the Farinograph after 30 min mixing andmixing continued for additional 5 min and 15 min, respectively, the patterns of the SDSbuffer extracts of the residues from these doughs still showed only faint bands of tripletzone proteins as obtained for the control dough [Fig. leA), lane (e)]. Thus, additionalmixing with iodate of an already mixed control dough did not result in recovery of tripletzone proteins. Furthermore, the presence of iodate during the extraction of a floursample with acetic acid did not affect protein extractability or electrophoretic patternsof the SDS buffer extracts of the residue fractions. These results suggest that the' iodateeffect' occurs only during dough mixing.

The results presented in Fig. 1 were for extracts of residue samples adjusted to containequal amounts of protein. When the mixing-induced decrease in protein content ofdough residues was not taken into account in the extraction with SDS buffer, thepatterns of residues from untreated doughs showed a slightly greater decrease in theintensity of triplet zone proteins than those shown in Fig. leA). Under these conditions,however, the triplet zone proteins extracted from residues of iodate-treated doughsshowed no significant variation related to mixing. Also, when freeze-dried iodate-treateddoughs were extracted directly with SDS buffer, the resulting patterns of the extractsshowed no differences in the triplet zone (results not shown). From these observationswe concluded that iodate stabilizes triplet zone proteins, preventing their breakdownand/or association with other dough constituents during miv"mg. In view of thesuggested stabilizing effect of iodate on triplet zone proteins and the observed decreasingprotein content of dough residues (Table I), the increase in intensity of triplet zoneproteins as shown in Fig. l(B) could be explained on the basis of an increase of tripletzone proteins relative to other proteins in the residues with increasing mixing time. Thus,when residue samples containing equal amounts of protein were extracted, the relativeproportion of triplet zone proteins in the extracts increased. This concentration effectmight also account for the appearance of additional bands in patterns of extracts ofresidues of doughs which had been subjected to prolonged mixing with iodate [Fig. I(B)and (C)]. Presumably, these additional proteins were also present in the residue of the:flour sample. However, their concentration probably was too low to give distinct bandsin the pattern of the SDS buffer extract.

The commonly proposed mechanism of the iodate effect in dough is the inhibition of

RESIDUE PROTEIN AND DOUGH MIXING 249

disulfide/sulfhydryl interchange reactions via oxidation of accessible thiol groups20-25.

Therefore, the results obtained in this study suggest that triplet zone proteins areinvolved in the interchange reactions during mixing.

To elucidate further the involvement of thiol-mediated interchange reactions in theeffect of dough mixing on triplet zone proteins, we examined the changes of theseproteins during mixing in the presence of the thiol-blocking agent N-ethylmaleimide(NEMI). The results complemented those for iodate-treated doughs. Dough mixing withNEMI induced an increase in the staining intensity of triplet zone proteins extractedfrom dough residues (results not shown).

Doughs of the water-extracted flour. It has been suggested that water-soluble flourcomponents are involved in the effect of iodate in dough26.27. Those results, togetherwith our findings suggesting that KI03 stabilized triplet zone proteins, seem to indicatethat the decrease in intensity of triplet zone proteins is related to their interaction withwater-soluble flour components. To address this possibility, the effect of mixing ontriplet band proteins in doughs from water-extracted flour was investigated. Inagreement with other reports13• 26, 28, dough from water-extracted flour did not show anybreakdown during extended mixing in the Farinograph. In contrast to results for controldoughs, electrophoretic pattern of SDS buffer extracts of the acetic acid-insolubleresidues of doughs from water-extracted flour showed that the triplet zone bands werenot affected by mixing (results not shown). This observation supports our hypothesisthat the events in the triplet band region during mixing were mediated through water­soluble components of the flour.

SDS-PAGE of extracts prepared under reducing conditions

Control doughs and doughs mixed in the presence of KI0 3 and NEMI. Electrophoreticpatterns of SDS buffer extracts, prepared from control and iodate-treated doughs underreducing conditions, were not affected by mixing (results not shown). Patterns ofextracts from acetic acid-insoluble residues of control and iodate-treated doughs,however, showed a decrease in band intensity of the HMW and LMW glutenin subunitswith increasing mixing time [Fig. 2(A) and (B), RI and R3]. Similar results were obtainedfor NEMI-treated doughs. These observations are consistent with the explanation thatthe increased solubilization of proteins during mixing results from an increasedextractability of glutenin. The decrease in intensity of the glutenin subuni ts was evidentafter I min mixing. Since all residues extracted with SDS buffer (with mercaptoethanol)contained equal amounts of protein, the decreasing proportion of glutenin in theresidues may be compensated by increasing relative proportions of other residue proteincomponents, for example aM, ~- and y-gliadins [Fig. I(A) and (B)]. Under reducingconditions, however, it was not possible to determine unequivocally if the relativeproportion of aM, p- and y-gliadins [Fig. 2(A) and (B), R4] in the residue increased withmixing. This might be caused by overlapping of gliadin bands and the bands of LMWglutenin subunits.

After sequential extraction of flour and doughs with acetic acid and SDS buffer(without mercaptoethanol), there was still a substantial amount of protein in the

250 D. SIEVERT EY AL.

RI

R2

R3

R4

R5

a b

(A)b e' d •

(B)

- 45

- 29

- 20-1

- 14-:3,

FIGURE 2. SDS-PAGE of SDS buffer extracts prepared under reducing conditionsof acetic acid-insoluble residues from control doughs (A) and iodate-treated doughs(B). Lanes (a)-(e), see Fig. 1. RI-R5, see text. Arrowheads indicate molecular weightregions of triplet band subunits: 52-56k and 22-23k. (f) Denotes reduced proteinextract of Neepawa flour used as a secondary standard; (g) denotes reduced marker

proteins. Electrophoresis was performed under normal conditions.

residues, which was extractable under reducing conditions (Fig. 3). In the extraction ofthe residues with SDS buffer containing mercaptoethanol, the sample size was notadjusted to a constant amount of protein. Electrophoretic patterns of control doughsshowed little change with mixing; the band intensities of glutenin subunits [Fig. 3(A), Rland R3] and a-, ~-, y-gliadins [Fig. 3(A), R4] appeared to decrease slightly. Patterns ofiodate-treated doughs, on the other hand, showed a greater decrease in intensity of theregions comprising HMW and LMW glutenin [Fig. 3(B), Rl, R3]. Electrophoreticpatterns of analogous extracts from NEMI-treated doughs were essentially the same asthose obtained for iodate-treated doughs. These results suggest a higher degree ofdisaggregation and/or depolymerization of glutenin, and hence its increased solubilityin acetic acid and SDS buffer without mercaptoethanol, when doughs are mixed in thepresence of iodate or NEMI.

Doughs of the water-extracted flour. In contrast to electrophoretic patterns of SDS

RI

R2

R3

R4

RS

RESIDUE PROTEIN AND DOUGH MIXING

- 20~

-116

- 97·4

- 66

- 29

- 20,1

251

a b C

CAl

d • a b c' d •

(8 )

FIGURE 3. SDS-PAGE of SDS buffer extracts prepared from residues underreducing conditions. Residues were obtained after sequential extraction of controldoughs (A) and iodate-treated doughs (B) with acetic acid and SDS buffer (withoutmercaptoethanol). Lanes (a)-(g), see Fig. 2. Rl-R5, see text. Arrowheads indicatemolecular weight regions of triplet band subunits: 52-56k and 22-23k. Electrophoresis

was performed under normal conditions.

buffer extracts of acetic acid-insoluble residues from control doughs [Fig. 2(A)], thepatterns of analogous extracts of residues from doughs of the water-extracted flourshowed no decrease in intensity of glutenin subunits (results not shown). These results,together with the observation that the acetic acid residues of water-extracted flour andits dough contained the same amount of protein, indicated that no solubilization ofproteins (glutenin) occurred when dough was mixed in the absence of the water solubles.This might be explained by the lack of development of a viscoelastic gluten network.Yoshida and Danno29 recently reported that the partial removal of gliadin by waterextraction procedures prevents formation of a viscoelastic gluten mass and therebyprecludes development and breakdown of dough during mixing.

Subunits of triplet band proteins. Subunits of triplet band proteins appear in theM r 52-58k and 22-23k regions of SDS-PAGE patterns under reducing conditions12, 15.

Since other protein bands are also resolved in these regions, it was not possible in our

252 D. SIEVERT ET AL.

RI

R2

R3

R4

R5

k

- 116

- 66

- 45'

- 29

- 14'3

a b c d e 9

FlGURE 4. SDS-PAGE of acetic acid-soluble slot protein from Neepawa flour andcontrol doughs. Lanes (a)-(g), see Fig. 2. Rl-R5, see text. Electrophoresis was

performed under normal conditions.

study to verify the absence or presence of triplet band subunits in patterns of extractsof residues obtained under reducing conditions (Figs 2 and 3, arrow-heads) or inpatterns of acetic acid and SDS buffer extracts prepared under non-reducing conditionsfollowed by addition of mercaptoethanol (not shown). Therefore, no conclusion can bemade as to whether the proportion of triplet band proteins, which would account for thedecrease in intensity in the patterns as shown in Fig. I(A), was present as breakdownproducts and/or in aggregated form with other proteins in the acetic acid extract, theSDS buffer extract of the acetic acid insoluble residue, or the residue remaining aftersequential acetic acid and SDS buffer extraction.

SDS-PAGE of' slot' protein

Control doughs. We investigated the effect of mixing on proteins that did not enter theseparating gel of SDS-PAGE under non-reducing conditions (' slot' protein) with a two­step one-dimensional electrophoretic procedure. SDS-PAGE analysis of reduced slotprotein of the acetic acid-soluble extracts from flour and doughs showed the presence ofglutenin subunits in a zone of slight background streaking (Fig. 4). Notably, the patternof slot protein of the acetic acid-soluble fraction was completely devoid of bands withmol. wts lower than approximately 35k. The subunit pattern in the 35-140k mol. wtregion (Fig. 4) was similar to the subunit patterns of glutenin obtained by several

Rl

R2

R3

R4

R5

RESIDUE PROTEIN AND DOUGH MIXING

k

-116

-66

- 29

- 14'3

253

a b c d e

FIGURE 5. SDS-PAGE of SDS buffer soluble slot protein of acetic acid-insolubleresidues from Neepawa flour and control doughs. Lanes (a)-(f), see Fig. 2. RI-R5,see text. Arrowheads denote bands with mol. wts of 90, 52, 22 and 18k. Electro-

phoresis was performed under normal conditions.

different fractionation and purification procedureslB.30-33. Mixing-related changes werenot detected in the pattern of slot protein of the acetic acid-soluble fractions of flour anddoughs of the cultivar Neepawa. Analogous slot protein of other cultivars were notinvestigated.

SDS-PAGE analysis of reduced slot proteins of SDS buffer extracts of acetic acid­insoluble residues (Fig. 5) gave patterns similar to those of the slot proteins of the aceticacid extracts (Fig. 4). However, some notable differences were observed. The patterns ofthe SDS buffer soluble slot proteins contained additional protein components at mol.wts of 90, 52, 22 and 18k (Fig. 5 arrow-heads).

The three hands with mol. wts 18, 22, and 90k were observed in the patterns of all thecultivars investigated. The 52k mol. wt band was also present in the patterns ofKatepwa, Glenlea and Kenya. In case of Pembina, Fielder, Augusta and Pitic,identification of the 52k mol. wt band was not possible because other bands weredominant in the 52k mol. wt region. The cultivars Kenya and Fielder showed anadditional band of 23k mol. wt. Notably, the 52, 23, and 22k mol. wt bands correspondin terms of mol. wt to subunits of triplet band proteins. Figure 5 indicates that theintensity of the band of 22k mol. wt declined with mixing, as was observed for the tripletbands [Fig. leA)]. The band of 18k mol. wt also showed a decrease in intensity.

254 D. SIEVERT ET AL.

k

- 97'4

- 66

- 45

- 29

- 14'3

o b c' d e 9

FIGURE 6. SDS-PAGE of SDS buffer soluble slot proteins of acetic acid-insolubleresidues from Neepawa flour and iodate-treated doughs. Lanes (a)-(g), see Fig. 2.RI-R5, see text. Arrowheads denote bands with mol. wts of 90, 52, 22 and 18k.

Electrophoresis was perfonned under nonnal conditions.

SDS buffer soluble slot protein of residues from all other wheat cultivars examinedgave a consistently similar response to dough mixing. With increasing mixing time thebands of 22 and 18k mol. wt gradually became fainter. The additional 23k mol. wt bandof the cultivars Kenya and Fielder followed the same pattern.

Doughs mixed in the presence of KIOa and NEMI. The two-step one-dimensionalelectrophoretic analysis of acetic acid-soluble slot protein of Neepawa doughs mixedwith iodate or NEMI gave patterns similar to those of analogous slot protein fractionsof control doughs. Slot protein of SOS buffer extracts from residues of iodate-treateddoughs, however, showed significant differences compared with analogous slot proteinfractions from control doughs. Mixing with iodate produced an increase in stainingintensity of the bands of 52, 22 and 18k mol. wt, which coincided with a slight increasein intensity of the overall pattern (Fig. 6). Mixing with NEMI gave similar results (notshown). As observed for control doughs, the change in intensity of the 22k mol. wt bandthat corresponded to triplet band subunits paralleled the changes in intensity of tripletband proteins [Fig. l(B)].

RESIDUE PROTEIN AND DOUGH MIXING 255

Analysis of slot protein of SOS buffer extracts of residues from all seven wheatcultivars confirmed an increase in intensity of the 22k mol. wt band when mixing wascarried out in the presence of iodate. Slot protein of the cultivars Kenya and Fieldershowed an increase of both, the bands of 22 and 23k mol. wt.

General Discussion

This study has shown that proteins, resolved in SDS-PAGE patterns under non­reducing conditions in the triplet band region (triplet zone proteins), respond similarlyto dough mixing. The mechanism of the modification of the triplet zone proteins duringmixing appears to involve sulfhydryl-disulfide interchange reactions. This mechanism isconsistent with the concept of disulfide interchange between LMW sulfhydryl (thiol)groups and HMW proteins, which has been proposed to account, in part, formodifications of polymeric wheat proteins during mixing (Bloksma and Bushuk34 andreferences cited therein). Since the reaction of iodate and NEMI in dough appears toproceed via oxidation and blocking of accessible thiol groups20-25, the increase in bandintensity of triplet zone proteins in the presence of these agents might be due toinhibition of disulfide interchange reactions. Conversely, the decrease in the bandintensity of the triplet zone proteins during mixing of control doughs could be explainedon the basis of reduction reactions that take place during thiol/disulfide interchangereactions. Since mixing of doughs from water-extracted flour gave no change in stainingintensity of triplet zone proteins, it is tempting to attribute the changes of these proteinsto interactions with components in the water soluble fraction of the flour. Based on theresults presented here, however, it is not possible to conclude whether the mixing-relateddecrease in band staining intensity of the triplet zone proteins in SDS-PAGE patternsof dough residue extracts was due to solubilization reactions or to association of tripletzone polypeptides with other constituents of the residue.

A role for triplet proteins in breadmaking has been speculated15 • 19 . The presence oftriplet zone proteins in the acetic acid-insoluble residue fractions and their extractabilityfrom the residues with SOS buffer (a relatively strong dissociating agent) under non­reducing conditions indicate that these proteins are associated with each other and/orwith other constituents of the residues by relatively strong non-covalent forces. In thepresent study, we detected-after reduction-subunits in SDS buffer extractable slotprotein of acetic acid-insoluble residues that corresponded in their mol. wts to the 52,23 and 22k mol. wt subunits of triplet band proteins. If, in fact, these proteins aresubunits of triplet band proteins, then it follows that at least a part of triplet bandproteins interact strongly with SDS buffer-extractable glutenin. This complex, however,does not enter the separating gel in SDS-PAGE unless the disulfide bonds of gluteninand/or triplet band proteins are fully reduced. In this context, it is interesting to notethat the mixing-induced changes in staining intensity of the 23 and 22k mol. wt bands,and in part, the 52k mol. wt band in SDS-PAGE patterns of the reduced slot proteinparalleled the observed changes in band intensity of triplet band proteins in patterns ofunreduced SDS buffer extracts of residue fractions. Singh et aZ,u reported that tripletband proteins are present along with glutenin in the peak I fraction of unreduced proteinextracts separated by size-exclusion high-performance liquid chromatography. Whether

256 D. SIEVERT ET AL.

their findings, together with our observations, can be related to aggregation of tripletband proteins with glutenin in residue fractions and to rheological properties such asdough strength, remains to be determined.

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