A Component of Wheat Flour Globulin Polymerized at Alkaline Sides and Depolymerized by Reduction Reversibly

Size: px

Start display at page:

Download "A Component of Wheat Flour Globulin Polymerized at Alkaline Sides and Depolymerized by Reduction Reversibly"

Susanna Hodge
5 years ago
Views:

1 Agric. Biol. Chem., 42 (7), 1397 `1402, 1978 A Component of Wheat Flour Globulin Polymerized at Alkaline Sides and Depolymerized by Reduction Reversibly Masaki TERADA, Junichi MINAMI and Takehiko YAMAMOTO*' General Research Laboratory, Nissin Food Products Co., Ltd., Ritto-cho, Shiga 525, Japan Received February 16, The SDS-disc electrophoretical analysis of wheat flour globulin revealed that upon incubation with "Kansui," a mixture of alkali carbonates, one (No. 9) of the components disappeared and another component (No. 11) increased significantly whose molecular weight was estimated to be more than one million. Incubation of flour globulin with "Kansui" resulted in decrease of SH groups of the globulin. 2. However, a mild reduction of the globulin incubated with "Kansui" caused reap pearance of component No. 9 and a decrease of component No Previous treatment of flour globulin with PCMB or AN completely inhibited such effects of "Kansui" as described above. 4. A fraction containing component No. 9 of flour globulin which was the main com ponent responsible to polymerization by "Kansui," was isolated from "Kansui"-treated globulin by chromatography on columns of Sephadex G200 and Sepharose 4B. 5. The component protein thus isolated showed a typical phenomenon of reversibility in depolymerization to the original component corresponding to No. 9 component by chemical reduction and polymerization to a component corresponding to No. 9 at alkaline sides. Our previous paper1) reported that "Kansui," a mixture of sodium and pottasium carbonates which is used for making a kind of Chinese noodle, affected wheat flour protein, especially the so-called globulin fraction, to bring a significant change in the disc electrophoretic pattern. The present paper describes the charac teristic phenomenon of wheat flour globulin caused by "Kansui" is attributed to a certain component which is polymerized at alkaline sides and depolymerized by reduction at neutra or slightly acidic sides, reversibly. MATERIALS AND METHODS 1. Preparation of * wheat flour globulin. Three hundreds g of wheat flour (Dark hard winter species) were suspended in 1500 ml of 0.2 % sodium chloride solution and the slurry was incubated at 40 C with *1 Faculty of Science, Osaka City University, Sumiyoshi-ku, Osaka. Abbreviations: SDS, sodium dodecyl sulfate PCMB, p-chloromercuribenzoate; AN, acrylonitril DTT, dithiothreitol; 2-ME, 2-mercaptoethanol. gentle stirring. Two hr later, the slurry was centri fuged and solid ammonium sulfate was added to the supernatant at 0.5 saturation and the mixture was allowed to stand overnight in the cold. The resulting precipitate was collected by centrifugation, dissolved in 0.2% sodium chloride, and extensively dialyzed against the same solution. The dialyzed solution was used as flour globulin. The protein concentration was usually 0.38%. when determined by the method of Kjeldahl. Protein concentration was sometime expressed by only absorbance at 280 nm. 2. Treatment of flour globulin with "Kansui." A 10% solution of "Kansui" which consisted of 60 K2C03, 36% Na2CO3 and 4 % Na2HPO4.7H20, was added to the globulin solution obtained above to bring the ph at 9.5, and the mixture was incubated under various conditions. The globulin solution thus treated was used as "Kansui"-treated globulin. 3. Determination of SH groups. Sulfhydryl groups were measured by the method of Ellmann.2) To estimate S-S linkages, on the other hand, the globulin solution was reduced with 0.1 % DTT at ph 9.0 and at 30 C for 2 hr in the presence of 0.1 % SDS. The mixture was then extensively dialyzed*2 against a solu- *2 Neither DTT nor Cl- was detected after 48 hr dialysis against five times changed water of a thousandtimes volume previously purged with nitrogen gas.

2 1398 M. TERADA, J. MINAMI and T. YAMAMOTO tion containing 0.2% sodium chloride and 0.1 % SDS at ph 6.0 and at 4 Ž, and subjected to estimation of total SH groups by the method of Ellmann. The globulin solution examined, if necessary, was concen trated in a membrane bag (Diafilter G-10T, Bioengi neering Co., Ltd., Japan) under nitrogen gas of 4.0 kg/ cm2 in the cold. The content of S-S groups was ob tained by subtracting free SH groups from total SH ones. 4. SDS-disc electrophoresis. The disc electro phoresis in the presence of SDS was carried out according to the method of Osborn.') Protein bands on the disc were stained with 0.5% Amido Black lob in 20% acetic acid, and the quantity of protein at each band was measured with a densitometer, Densitorol of flour globulin with no "Kansui," disap peared almost completely by incubation with "Kansui." Some other components such as No. 4, 5 and 10 also decreased, but their degrees were slight. Component No. 3, on the other hand, slightly increased. The change of SDS-disc electrophoretic pattern of flour globulin caused by "Kansui," however, was not observed when flour globulin (0.38%) was previously treated with PCMB (2.1 mm) as a SH blocker, as shown in Fig. 2. Treatment with AN (3.3 mm, ph 6.0, 30 Ž, 1 hr) gave a similar effect to that by PCMB. DMU-2, Toyo Kagaku Co., Ltd., Japan. RESULTS 1. Change in SDS-disc electrophoretic pattern of the globulin by treatment with "Kansui" The SDS-disc electrophoretic patterns of the globulin were changed significantly by incuba tion with "Kansui." As presented in Fig. 1, a component symbolized as No. 9 in the pattern FIG. 2. Effect of p-chloromercuribenzoate (PCMB) and Acrylonitril (AN) on SDS-Disc Electrophoretic Patterns of Flour Globulin. (a), treated with PCMB, and incubated at ph 5.8, 40 Ž for 2 hr; (b), treated with PCMB and incubated at ph 9.5, 40 Ž for 2 hr; (c), treated with AN, and incubated at ph 5.8, 40 Ž for 2 hr; (d), treated with AN, and incubated at ph 9.5, 40 Ž for 2 hr. 2. Changes in contents of SH and S-S groups by treatment with "Kansui" FIG. 1. Changes in SDS-Disc Electrophoretic Pro perties of Flour Globulin Incubated with or without "Kansui." (a), before incubation; (b), after incubation for 4 hr without "Kansui"; (c), after incubation for 0.5 hr with "Kansui"; (d), after incubation for 2 hr with "Kansui." As shown in Fig. 3, SH groups of flour globulin whose content was about 13,moles per g, remained unchanged when incubated at ph 5.8. However, on incubation with "Kan sui," ph 9.5, the SH groups rapidly decreased to a level of 3 to 4,ƒÊmoles per g, and remained at a const level thereafter.

3 A Flour Globulin Component Polymerized and Depolymerized Reversibly 1399 The change in content of S-S groups was not so clear as that of SH groups between globulin incubated with and without "Kansui," because the content of S-S groups was much greater than that of SH groups originally. FIG. 4. Changes in SDS-Disc Electrophoretic Patterns by Reduction with 2-Mercaptoethanol (2-ME) FIG. 3. Changes in SH and S-S Contents of Flour Globulin Caused by "Kansui." O-O, incubated without "Kansui" (ph 5.8, 40 Ž); i-e, incubated with "Kansui" (ph 9.5, 40 Ž). 3. Reduction of "Kansui"-treated globulin with 2-ME The flour globulin, 0.7 mg, incubated with "Kansui" (ph 9.5) at 40 Ž for 1 hr was reduced with 0.42 ml of several concentrations of 2-ME at ph 7.2 and at 50 Ž for 2 hr in the presence of SDS, and their SDS-disc electrophoretic patterns were investigated. As shown in Fig. 4, reduction of the "Kansui"-treated glo bulin by a proper concentration of 2-ME decreased component No. 11 and increased com ponent No. 9 (Fig. 4c). However, in this case, component No. 6 was decreased, though it was of Globulin Fraction Treated with "Kansui" for 1 hr. (a), reduced with 0.68 ƒêm 2-ME; (b), reduced with 68 ƒêm 2-ME; (c), reduced with 0.68 M 2-ME; (d), reduced with 0.68 M 2-ME, ("Kansui," untreated globulin fraction as the control), cipitated with ammonium sulfate of concen trations between 0.25 and 0.5 saturation was used, because this fraction was found to contain the principal component responsible to poly merization with "Kansui." The flour globulin solution (11.4 mg in 5 ml) incubated with "Kansui" at ph 9.5 and at 40 Ž for 1 hr was neutralized to ph 7.0, was applied to a column of Sephadex G-200(1.8 ~ 37.5 cm) equillibrated with 2 ~ 10-2 M phosphate buffer, ph 7.0, and eluted with the same buffer solution. The elution pattern is shown in Fig. 5 comparing not affected on incubation with "Kansui." Reduction of "Kansui"-treated globulin to a slightly excessive extent, resulted in the com plete disappearance of component No. 11 and reappearance of No. 9. Several other com ponents, however, were also influenced under the reduction condition and No. 6 component was decreased while No. 7 and 8 components were slightly increased, though they were not affected by incubation with "Kansui" originally. 4. Fractionation of "Kansui"-treated globulin In this experiment the flour globulin pre- FIG. 5. Gel Filtration (Column, 1.8 x 37.5 cm of Sephadex G200) of Globulin Fraction Treated with or without "Kansui." -*, with "Kansui"; o-o, without "Kansui"; Yo, indicates the void volume of the column.

4 1400 M. TERADA, J. MINAMi and T. YAMAMGTO with that obtained from flour globulin without previous incubation with "Kansui" as the con trol. Figure 5 shows that flour globulin treated with "Kansui" greatly decreased in peak b _ and increased in peak a compared with the control. The molecular weight of protein of peak a was considerably larger than that of peak b. As for peak c whose mole cular weight was presumed to be less than peak b, no appreciable change in amount was observed by treatment with "Kansui." Peak a' (30 mg in 30 ml) obtained from "Kansui" -treated globulin as in the exeriment of Fig. 5 was concentrated using a membrane bag and subjected to purification by a column of Sepharose 4B (2.5 ~ 65 cm) buffered with 2 ~ 10-2 M phosphate, ph 7.0, and the eluate was fractionated into three fractions as shown in Fig. 6. FIG. 7. SDS-Disc Electrophoresis of Peak 1, 2 and 3 in Fig. 6 after Reduction with Dithiothreitol (DTT). (a) peak 1, (b) peak 2, and (c) peak 3 of Fig. 6 respectively. globulin untreated with "Kansui." Fraction 3, on the other hand, was quite different in the FIG. 6. Chromatography of Peak a' Fraction on a Column of Sepharose 4B. Peak a' fraction in Fig. 5, 30 ml, 2.14 at 280 nm per cm, was concentrated, applied to the column, and fractionated. 5. SDS-disc electrophoretic patterns of the globulin fractions isolated by the column chromatographies An aliquot of each peak fraction separated and concentrated in the experiment of Fig. 6 (1.0 mg/ml) was reduced with 0.1 ml of 54 mm of DTT under the conditions described in MATERIALS AND METHODS, and subjected to SDS-disc electrophoresis. The results are presented in Fig. 7, showing that fraction 1 and 2 of Fig. 6 gave similar patterns in the SDSdisc electrophoresis with the peaks corresponding to some of components shown by the pattern from the former two and the peaks shown were not matter in the present study. A portion of fraction 2 of Fig. 6 was concen trated by ultrafiltration, reduced by DTT of 23 mm, and dialyzed against 2 x 10-3 M phos phate buffer, ph 6.0, containing 1 x 10-3M EDTA. The SDS-disc electrophoretic pattern of the fraction thus reduced was investigated with and without incubation with "Kansui." The results shown in Fig. 8a and b indicate that the fraction before incubation with "Kansui," gave only a few of the components shown by "Kansui"-untreated globulin. On the other hand, the fraction lost the components numbered as 5, 8 and 9, especially No. 9, and increased component No. 11 by incubation with "Kansui," ph 9.5. The solution of frac tion 2 once incubated with "Kansui," was again reduced with 23 mm DTT in the pre sence of 8 M urea and then incubated with "Kansui." The electrophoretic patterns are presented in Fig. 8c and d, showing that the change in electrophoretic pattern was com pletely reversible. However, the change in electrophoretic pattern of the fraction caused

$(a), peak 2 fraction reduced; (b), after incubating a at ph 9.5, 40 Ž, 1 hr; (c), (b) was reduced with DTT in the presence of urea, and dialyzed; (d), (c) was incubated at ph 9.5, 40 Ž, 1 hr. by incubation with "Kansui" was not observed with the samples previously incubated with SDS (0.$

5 A Flour Globulin Component Polymerized and Depolymerized Reversibly 1401 FIG. 8. Reversibility of SDS-Disc Electrophoretic Pattern Shown by Peak 2 in Fig. 6. (a), peak 2 fraction reduced; (b), after incubating a at ph 9.5, 40 Ž, 1 hr; (c), (b) was reduced with DTT in the presence of urea, and dialyzed; (d), (c) was incubated at ph 9.5, 40 Ž, 1 hr. by incubation with "Kansui" was not observed with the samples previously incubated with SDS (0.95 f). DISCUSSION The observation that a decrease in content of SH groups of flour globulin caused by in cubation with "Kansui" was remarkable at initial of the incubation to readily arrive at a certain low level (Fig. 3), was consistent with the finding that the change in SDS-disc electro phoretic pattern of flour globulin occurred in only 30 min of incubation with "Kansui," and no more striking changes were observed thereafter. Therefore, the decrease of SH content was considered to correlate with change in SDS-disc electrophoretic pattern. The most striking change in electrophoretic pattern caused by "Kansui" was disappearance of a component symbolized No. 9 and ap pearance or increase of another component No. 11, shown in Fig. 1. The change of SDSdisc electrophoretic pattern by "Kansui" was independent on concentration of flour globulin. But the change was inhibited by previous treat ment of globulin with PCMB or AN (Fig. 2). Also, a mild reduction of "Kansui"-treated globulin reversed the pattern to be almost similar to that shown by "Kansui"-untreated globulin. No. 11 component was estimated to have a molecular weight of more than one million based on the elution profile through a column of Sephadex G-200. A fraction containing No. 11 component was obtained from "Kan sui"-treated globulin by chromatography using columns of Sephadex G-200 and Sepharose 4B. The globulin employed in the experiment was that fractionally precipitated between 0.25 and 0.5 saturation of ammonium sulfate. The fraction obtained was shown to consist of four components when it was reduced (Fig. 7). On the other hand, the "Kansui"-treated glo bulin was completely reversed by a mild reduc tion for the two electrophoretical components, No. 9 and 11 (Fig. 4c). Thus the component No. 9 was found to be responsible to polymeri zation to form No. 11 that occurred at alkaline sides, as can be seen from Fig. 8. The mole cular weight of component No. 9 was estimated to be about 7.7 x 104. Thus, it follows that some decade molecules of component No. 9, a SH protein, was polymerized at alkaline sides to give component No. 11 whose molecular weight was more than a million with a high content of S-S group. The pattern of Fig. 4c, however, somewhat differedfrom that shown by "Kansu i"- incubated globulin. The difference was attributed to that resulted by reduction of several other components, especially No. 6. But, those components were considered to be out of the present discussion, because they were not affected by incubation with "Kansui," originally. It may be clear that the polymerization de scribed above was resulted by oxidation that occurred at alkaline sides of ph values around 9.5, because the polymerization product was readily reversed to the original SH protein by reduction, though a further study will be necessary to elucidate the mechanism underly ing the polymerization phenomenon. The contents of S-S and SH groups of com ponent No. 9 were not examined, because the component obtained was insufficient in amount for investigation. However, those of the frac tion of a' obtained in Fig. 5 were investigated

6 1402 M. TERADA, J. MINAMI and T. YAMAMOTO after reduction and dialysis against 2 x 10-2 M phosphate buffer (ph 6.0) containing I mm EDTA. The contents of S-S and SH groups of the fraction a' reduced were estimated to be 7.1 and 78.0 moles per g protein, respectively. By incubation with "Kansui," the SH content rapidly decreased to a level of 15.0,ƒÊmoles per g protein, though it was obscure whether these values were meaningful, because the fraction a' was isolated from flour globulin incubated with "Kansui" and then reduced. Nevertheless, unlike the original globulin, the fraction a' had a high content of SH group as compared with S-S groups. It is known that rheological properties of dough are readily modified by changing ph to alkaline sides as the case by adding "Kan sui." Recently, we reported 4 that the effect of "Kansui" was inhibited by pretreatment of dough with PCMB. This phenomenon was similarto that caused by bromates or iodates,5 `11) Thus the principal effect of "Kansui" on rheological properties of dough may be attributed to oxidation of SH groups and/or interchange reaction between SH and S-S. The main protein of wheat flour is the so-called gluten. However, in the experiment performed inde pendently, gluten was not shown to receive any remarkable effect of "Kansui" on the SDS-disc electrophoretic pattern. On the other hand, globulin fradtion was significantly influenced by "Kansui," and thus the component of globu lin fraction described above may be most res ponsible to the change of Theological properties of dough caused by "Kansui." However, more study will be necessary to solve the, problem, because the amount of the component in question seems to be too small to explain the "Kansui" effect discussed for long. REFERENCES 1) M. Terada, J. Minami and T. Yamamoto, Agric. Biol. Chem., 41, 23 (1977). 2) G. L. Ellmann, Arch. Biochem. Biophys., 82, 70 (1959). 3) K. Weber and M. Osborn, J. Biol. Chem., 244, 4406 (1969). 4) M. Terada, J. Minami and T. Yamamoto, Agric. Biol. Chem., 42, 365 (1978) 5) H. Matsumoto and I. Hlynka, Cereal Chem., 36, 513 (1959). 6) W. Bushuk, ibid., 38, 438 (1961). 7) B. Sullivan, L. K. Dahle and J. H. Schipke, ibid., 40, 515 (1963). 8) D. E. Smith and J. D. Mullen, ibid., 42,275 (1965). 9) B. Belderok, Getreide Mehl, 17, 20 (1967). 10) A. H. Bloksma, Cereal Chem., 49,104 (1972). 11) K. Tanaka and. W Bushuk, ibid., 50, 590 (1973).

Biochemical Studies on the Mineral Components in Sake Yeast. Part V. The Relationship of the Mineral Composition of Yeast to Fermentation

[Agr, Biol. Chem. Vol. 30, No. 9, p. 925 `930, 1966] Biochemical Studies on the Mineral Components in Sake Yeast Part V. The Relationship of the Mineral Composition of Yeast to Fermentation By Tsuyoshi