Direct molecular weight determination in the evaluation of dissolution methods for unreduced glutenin

Journal of Cereal Science 39 (2004) 1–8 www.elsevier.com/locate/jnlabr/yjcrs

Direct molecular weight determination in the evaluation of dissolution methods for unreduced glutenin C. Arfvidssona, K.-G. Wahlunda,*, A.-C. Eliassonb a

Department of Technical Analytical Chemistry, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden b Department of Food Technology, Center for Chemistry and Chemical Engineering, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden Received 15 January 2003; revised 16 April 2003; accepted 8 May 2003

Abstract A comparison of two dissolution methods, gentle stirring and sonication, for solubilising the ultra-large wheat protein glutenin was made based on a direct determination of the molecular weight of dissolved glutenin. The possible physical breakdown of glutenin by sonication was evaluated. Glutenin was isolated by extraction with hydrochloric acid and freeze dried. The extracted glutenin was dissolved either by gentle stirring at 4 8C in phosphate buffer pH 6.8 containing sodium dodecyl sulphate (SDS) or by ultrasonication in the same SDS containing buffer immersed in an ice-bath. The weight average molecular weight and the molecular weight distribution of the dissolved material were determined using asymmetrical flow field-flow factionation using a multiangle light scattering detector coupled on-line with a refractive index detector. These determinations showed that sonication caused physical breakdown of the ultra-large glutenin proteins. The average molecular weight was found to be 5 £ 106 after sonication and 2 £ 107 after gentle stirring. q 2003 Elsevier Ltd. All rights reserved. Keywords: Molecular weight; Glutenin; Field-flow fractionation; Multiangle light scattering

1. Introduction Glutenin comprises large molecules that are formed from polypeptide chains linked by disulphide bonds (Eliasson, 1993; MacRitchie, 1992) and are thought to have molecular weights ranging from a few hundred thousand to many millions. Due to the low solubility of glutenin in aqueous media vigorous dissolution methods are required to bring it into solution. The solubilisation of total flour proteins without reduction of the disulphide bonds has been investigated (Bottomley et al., 1982; Danno, 1981; Danno et al., 1974) and various solvents compared for the extraction of unreduced glutenin. These studies showed that solutions of sodium dodecyl sulphate (SDS) are the most Abbreviations: M, molecular weight; MD, molecular weight distribution; Mw , weight average molecular weight; FFF, field-flow fractionation; AsFlFFF, asymmetrical flow field-flow fractionation; rms, radii, root-mean-square radii; MALS, multiangle light scattering; RI, refractive index; SDS, sodium dodecyl sulphate; HPLC, high-performance liquid chromatography; SEC, size-exclusion chromatography. * Corresponding author. Tel.: þ 46-46-222-8316; fax: þ46-46-222-4525. E-mail address: [email protected] (K.G. Wahlund). 0733-5210/04/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0733-5210(03)00038-9

efficient solvents but that extractability varies between wheat flours (Bietz, 1984; Danno and Hoseney, 1982; Moonen et al., 1982). It has also been shown that the total protein in a very strong wheat flour was almost completely extracted, without reduction of disulfide bonds by applying mechanical shear with an ultrasonic probe in an SDS solution at pH 6.9 (Singh et al., 1990). Glutenin was dissolved in the presence of SDS using either gentle stirring or sonication. The increased solubility of flour proteins by sonication, compared to gentle stirring, has previously been explained by a reduction in size of glutenin I molecules (Moonen et al., 1982). It was concluded that sonication reduces the size of glutenin I polymers by breaking covalent bonds. Breakage of peptide bonds has also been suggested after prolonged sonication (Singh et al., 1990) but since disulphide bonds are weaker than peptide bonds (MacRitchie, 1975) sonication for as short as 15 s may result predominantly in cleavage of disulphide bonds (Weegels et al., 1994). In a more recent study (Morel et al., 2000), the effect of sonication time and intensity on size distribution and extractability was investigated.

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Molecular weight distributions are probably altered by extensive shear forces (Danno, 1991; MacRitchie, 1975; Weegels et al., 1994), such as sonication, but because it is difficult to measure the molecular weight of dissolved glutenin due to the lack of suitable methods for separation of these ultra-large molecules (Southan and MacRitchie, 1999) it has not been possible to assess the impact of sonication. Flow field-flow fractionation (FlFFF) has been used to fractionate ultra-high molecular weight glutenin proteins found in wheat flour (Stevenson and Preston, 1996, 1997; Wahlund et al., 1996). The principle of FFF has been reviewed (Giddings, 1993; Myers, 1997) and its theoretical and experimental basis described (Wahlund and Giddings, 1987; Litze´n et al., 1993; Giddings et al., 1992; Giddings et al., 1977). The method separates macromolecular species in a thin open separation channel according to differences in their diffusion coefficient. In the absence of a porous stationary phase and, consequently, an upper exclusion limit, the method has been useful in separations of ultra-large macromolecules where many of the most common separation methods fail to give the necessary resolution. The unique applicability of FlFFF connected on-line to MALS detection in the molecular weight and size determination of high molecular weight samples has been demonstrated (Thielking et al., 1995; Thielking and Kulicke, 1996; Wittgren and Wahlund, 1997) and the theoretical basis of the light scattering calculations described (Wyatt, 1993). Determination of the weight average molecular weight ðMw Þ and MD of ultra-large, non-reduced glutenin using asymmetrical flow FFF (AsFlFFF) connected on-line to MALS was recently introduced (Arvidsson, C., Wahlund, K.-G. and Eliasson, A.-C., unpublished data). In this study, the classical SDS dissolution of glutenin using gentle stirring for several days was applied in an attempt to avoid degradation, due to scission of covalent, disulphide bonds (Singh and MacRitchie, 1989). However, a residue still remained after 9 days of gentle stirring. The weight average molecular weight of the dissolved glutenin was 2 £ 107 : Recently, multiangle light scattering has also been used in combination with size-exclusion chromatography (SEC, SEHPLC) and reversed phase chromatography (RP-HPLC) to measure the molecular weight of glutenin and other proteins (Bean and Lookhart, 2001; Carceller and Aussenac, 2001; Lookhart, 1997). Due to the exclusion limit of the SEC columns all sample material of molecular weight higher than about 1 £ 107 is co-eluted in the exclusion volume (Jumel et al., 1996), limitting the determination of the molecular weight distribution. The upper molecular weight limit of glutenin can as a result cannot be accurately judged. On the contrary, with AsFlFFF– MALS even the highest molecular weight material can be separated and the complete MD can be obtained. The objective of the present study was to use the combination of AsFlFFF – MALS – RI to investigate

differences in the MD of dissolved glutenin after using two different dissolution methods, sonication and gentle stirring.

2. Experimental 2.1. Instrumental set-up The experimental set-up and procedures for AsFlFFF were the same as in previous studies (Andersson et al., 2001) with the MALS instrument (Dawn DSP laser photometer, Wyatt Technology, Santa Barbara, CA, USA), in combination with the interferometric refractometer (refractive index, RI, Optilab DSP, Wyatt Technology) connected on-line to the AsFlFFF channel. The MALS and RI instruments were calibrated before use with pure filtered toluene and standard solutions of sodium chloride in water of known RI increment, respectively, as recommended by the manufacturer. Normalization of detector angles other than the calibrated 908-angle was performed using an isotropic scatterer, bovine serum albumin (BSA), dissolved in the mobile phase. The interconnective volume between the two detectors was determined to allow matching of the light signal to obtain accurate molecular weight determinations. Jumpers were used on the amplifier booster PC board (Hardware Manual for the DAWN DSP Light scattering instrument, Wyatt Technology) to decrease the sensitivity of the light scattering detectors and keep their responses within range. Data collection and evaluation was performed using Astra software (Wyatt Technology, Santa Barbara, CA). The thickness, w; of the AsFlFFF channel was determined using ferritin (Litze´n, 1993) to 90 mm, giving a geometrical void volume of the channel of 0.315 ml. The lower channel wall was a NADIR UF 10-C regenerated cellulose ultrafiltration membrane (Hoechst AG, Wiesbaden, Germany) with a molecular weight cut-off of 10,000 was used. A 0.02 mm filter, Anodisc 25, Cat. no. 6809-6002 (Whatman International, Maidstone, UK) made of aluminium oxide with a pore size of 20 nm was inserted before the channel inlet. No in-line filter to the MALS detector was used. The channel was kept at an ambient temperature throughout the experiment. 2.2. Asymmetrical flow field-flow fractionation procedure The AsFlFFF analysis comprises three steps: the injection/focusing step, the elution step and the washing step. In the injection/focusing step 20 –200 ml of sample was injected using an HPLC pump (Kontron HPLC 422, Kontron Instruments, Milan, Italy) at a rate of 0.20– 0.50 ml/min. Another HPLC pump (Kontron HPLC 422) was used to deliver an inlet flow rate, Fin ; of 2.0 ml/min to the channel. After injection the sample was focused for another 0.5 min. The sample components were then eluted

C. Arfvidsson et al. / Journal of Cereal Science 39 (2004) 1–8

at a Fin of 5.2 ml/min. In the elution step the inlet flow rate was divided in the channel into an axial flow rate through the channel, Fout ; and a perpendicular cross flow rate, Fc : The ratio, Fc =Fout ; was set by restriction, using needle valves (Hoke Valve 1656 G2YA, Hoke Inc., Cresskill, NJ, USA), at the two outlets and varied between 5 and 10. In a final step a Fin of 5 ml/min washed the channel in the backward direction for 3 min. The switch in flow directions between the three analysis steps was performed by one two-way (Valco E C4W) and one four-way (Valco E-ST 4UV) motor driven valve (Valco Europe, Schenko, Switzerland). Kontron software (Gynkotek, Germering, Germany) was used to control the HPLC pumps as well as the motor driven valves. A pre-filtered (0.2 mm filter, RC-Vliesversta¨rkt Order no 18407-47N, Sartorius AG, Goettinger, Germany) and degassed 50 mM sodium phosphate, pH 6.8, 0.1% SDS was used as the carrier. 2.3. Dissolution of glutenin Ten protein fractions from the flour of Triticum aestivum L. ‘Mexico 8156’ (11.7% w/w protein) were prepared according to Lundh and MacRitchie (1987), by successive extractions of pre-isolated gluten protein with diluted HCl. The gluten protein was prepared (Lundh and MacRitchie, 1989) by hand-kneading a dough in distilled water after the non-starch lipid had been removed using the protocol of MacRitchie (1987). Gluten protein (35 g) was used for the extractions (Lundh and MacRitchie, 1989; MacRitchie, 1987). Fractions 1 – 3 were obtained with 0.625 mM HCl (800 ml), 2 –6 with 0.625 mM HCl (400 ml), and 7 –9 with 1.5 mM HCl (400 ml). The residue from the final extraction was dispersed in a small volume of water (fraction 10). After centrifugation the supernatant from each fraction was collected, the pH adjusted to 5.8, and the solution freezedried. The total amount of gluten extracted (expressed as percentage of the total protein in the sample) exceeded 85%. The percentages of protein in four different molecular weight ranges (large glutenins, glutenins, gliadins, albumins and globulins) for each fraction were also previously estimated, using the area of chromatograms from SEHPLC analyses (Lundh and MacRitchie, 1989). The results show that an increasing proportion of glutenin was extracted as the acid concentration increased. The proportion of glutenin in fraction 6 – 9 was estimated to about 75 –80% but only 2.5– 5% of the total gluten protein was estimated to be present in each of these fractions. Based on these experiments the freeze-dried extracted fraction 6 was chosen for further investigation. The freeze-dried fraction was dissolved using one of two glutenin dissolution methods, gentle stirring and sonication. Dissolution by gentle stirring was performed by mixing the freeze-dried sample with buffer containing 50 mM sodium phosphate buffer, pH 6.8 and 0.25% SDS for 3 –7 days at 4 8C. Only a minimum of mechanical action from a magnetic stirring bar, spinning at about 500 rpm, was

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applied during dissolution (samples 5 – 7 in Table 1). Fresh dissolution buffer was added to the undissolved material remaining after centrifugation of samples 5 and 6 at 6000 min1 for 30 min using a WIFUG 102 09 centrifuge (Chemico, Stockholm, Sweden). The dissolution procedure was then repeated for another 7 –9 days (with repeated stirring, samples 5 and 6 in Table 1). Dissolution by sonication was performed essentially as described previously (Wahlund et al., 1996) but with precautions (Morel et al., 2000; Singh et al., 1990) to prevent over-sonication. The freeze-dried sample was mixed on a vortex mixer with dissolution buffer for 10 s followed by a sonication (Sonifier B-12 with 3 mm diameter microtip probe, Branson Sonic Power Co., Danbury, CN, US) at one of four sonication intensities between 19 and 34 W described in Table 1 (samples 1– 4). The sonication was repeated in two to three steps of maximum 15 s duration. The sample tube was immersed in cold ice-water during the whole sonication. The mixture was finally centrifuged at 6000 min21 for 30 min using the WIFUG 102-09 centrifuge and the clear or slightly opalescent supernatant obtained placed at 6 8C awaiting analysis. Since none of the samples were completely dissolved by either gentle stirring or sonication the sample concentration could be determined only from the concentration (RI) signal in combination with the chosen dn=dc value. The injected sample concentration varied between 0.2 and 3 mg/ml taken as protein (exclusive of the adsorbed SDS). These concentrations are considered low enough to fulfil previously reported requirements (Folta-Stogniew and Williams, 1999; van Dijk and Smit, 2000; Wyatt, 1993) in order to Table 1 Dissolution of glutenin by sonication and by gentle stirring Dissolution Mass of Sonication samples freeze-dried power (W) fraction 6 in dissolution buffer (mg/ml)

Number Weight average of days molecular weighta of gentle ( £ 1027) stirring Average Range

Sonication 1 2 3 4

5.0 7.2 8.4 8.1

19 21 24 24– 35

0.6 0.4 0.6 0.5

0.5– 0.6 0.3– 0.4 0.6– 0.8 0.4– 0.6

Gentle stirring 5 46 6 45 7 47

3 5 7

2 3

2–3 2–3

Repeated stirringc 5 6

9 7

2 2

2–3 1–2

a

b

Triple analysis. Not determined. c Undissolved material (not quantified) from the dissolution by gentle stirring was subjected to repeated dissolution by stirring in 1.0 ml dissolution buffer. b

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assume the A2 c-term to be zero in the light scattering equation.

3. Results and discussion 3.1. Molecular weight determinations of dissolved glutenin The flour, Mexico 8156, has excellent bread making performance and yields strong doughs, which suggests that it contains significant amounts of glutenin. Previous studies on fractions of this flour have shown an increasing amount of glutenin with increasing concentration of acid extracant (Lundh and MacRitchie, 1989) and the presence of ultrahigh molecular weight glutenin components in fraction 8 as determined by AsFlFFF (Wahlund et al., 1996). This suggests that fraction 6 of Mexico 8156 also should contain significant amounts of glutenin. The molecular weights and the root-mean-square (rms) radii were determined from the MALS –RI data using the Berry formalism (Berry, 1966) with a first or second order fit in the Debye plot as suggested for high molecular weight components (Wyatt, 1993; Andersson et al., 2001). Where possible only the lowest light scattering angles (angle 26 – 908), those most important in the extrapolation to zero angle, were used. An accurate determination of the molecular weight by light scattering critically depends on the RI increment, dn= dc; used, since the optical constant in the light scattering equation is proportional to ðdn=dcÞ2 and thus the measured M is sensitive to errors with a dependence ðdn=dcÞ22 (Wyatt, 1993). However if the concentration is measured with an RI detector the product of the optical constant and the concentration depends linearly on dn=dc and the M dependence becomes ðdn=dcÞ21 instead. As a result any deviation of the value of dn=dc used, from the correct value will have less influence on the results. For standard proteins, such as BSA, in phosphate buffer pH 7, dn=dc values around 0.186 ml/g have previously been determined (Folta-Stogniew and Williams, 1999; van Dijk and Smit, 2000; Wen et al., 1996). In the presence of SDS the observed values of dn=dc for proteins are quite different (Kameyama et al., 1982; Miyake and Takagi, 1981; Takagi et al., 1980). Different SDS concentrations resulted in dn=dc values in the range of 0.28– 0.36, which suggested that the dn=dc varies with the amount of SDS bound to the protein. At about 1.4 g of SDS per gram of protein, shown previously to be the maximum amount of SDS bound (Reynolds and Tanford, 1970), the dn=dc was 0.32 – 0.34. Due to the binding of SDS to proteins the dn=dc has to be measured at equilibrium, which is established by dialysis against the buffer used (Miyake and Takagi, 1981; Eisenberg, 1976). The concentration of the protein, excluding the mass of bound SDS, also has to be known if the molecular weight of, not the complex in toto but the sole protein, is to be estimated (Miyake and Takagi, 1981). Using on-line dn=dc determination in SEC, the dialysis prior to the dn=dc measurement

could be omitted if the separation column had been thoroughly equilibrated with the buffer before use (Kameyama et al., 1982; Takagi et al., 1980). In a recent study on glutenin proteins by SEC –MALS (Bean and Lookhart, 2001) dn=dc values in the presence of 1% SDS were measured on-line (Astafieva et al., 1996). It was further assumed that all the injected protein was completely recovered from the column, that the protein was free of contaminants and that the protein concentrations were measured precisely. The reported dn=dc value of 0.32 ml/g for glutenin in the presence of SDS was however similar to those previously determined for other proteins in SDS. The main objective of this study was to investigate possible deviations in molecular weight and MD between glutenin dissolved by gentle stirring and glutenin dissolved by sonication. Since such deviations are unaffected by the dn=dc value used, the previously reported dn=dc values in the literature were reviewed and led to an estimate of dn=dc to 0.3 ml/g to be used in this investigation. No particular dn=dc measurements were therefore made. As mentioned above the use of a RI detector to determine the sample concentrations further decreased the influence of dn=dc on the results. It should be noted that the determination of the rms radius is independent of the input value for dn=dc; as it is obtained from the slope of the angular dependence (Wyatt, 1993). All molecular weights reported are considered apparent since it is not known to what degree glutenin existed as free molecules and non-covalent aggregates. A comparison was made between the classical SDS dissolution method with gentle stirring and the more recent method with SDS and sonication. To avoid over-sonication and unnecessary degradation of the glutenin, previous investigations (Singh et al., 1990; Morel et al., 2000), where effects of sonication time, sonication power and solid-to-solvent ratio are discussed, were taken into consideration. The impact of sonication on the MD of the ultra-large, non-reduced glutenin was as a result investigated at four different sonication powers (Sonication samples 1– 4 in Table 1). In no case was the sonication time greater than 15 s. None of the four sonicated samples were, as a consequence, completely dissolved. They all contained non-dissolved material when the sonication was terminated. This is probably due to the caution taken to avoid over-sonication, since protein extraction levels , 100% have been reported by others (Danno, 1981). As shown in Fig. 1A (sonication sample 1 in Table 1) the molecular weights determined by AsFlFFF – MALS – RI roughly spanned the range 105 – 107 for the high molecular weight fraction at 3.5 – 5.5 min and the weight average molecular weight was 5 £ 106 : The molecular weight of the highest molecular weight components of the sonicated solution was variable due to their low concentrations. Their contribution to the weight average molecular weight may still be significant due to their extremely high molecular weight. Since the Mw is estimated from a low range of scattering angles a sometimes out-of-range response at

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the very lowest angles may have decreased the precision in the Mw and contributed to the existing variation within the triplicate analysis (Table 1). No significant difference in determined weight average molecular weight between the four sonicated samples was observed (Table 1). The determined molecular weight ranges of glutenin dissolved by sonication using AsFlFFF– MALS– RI were similar to those determined by SEC – MALS (Bean and Lookhart, 2001; Carceller and Aussenac, 2001) but the upper size limit could not be determined accurately in the SEC studies due to the column exclusion limits. In comparing the results from sonication with those from gentle stirring (Table 1) using AsFlFFF, the weight average molecular weight was significantly lower in the samples dissolved by sonication. Comparing Fig. 1A with B it is also obvious that the distribution of molecular weights were different. The molecular weight range of the high molecular weight material in the sonicated sample was

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lower (105 – 107) than in the gently stirred sample (106 to above 108). This demonstrates that the glutenin has been partly degraded by the sonication procedure. That degradation was occurring was further supported by the fractogram shown in Fig. 1a from the analysis of glutenin dissolved using sonication power 1 that had a higher and more distinct low molecular weight fraction peak at 1.4 min than the fractogram from analysis of glutenin dissolved using gentle stirring (Fig. 1b). Due to the overloading phenomena discussed below, as well as different flow rate conditions, the low molecular weight fraction of glutenin was, after dissolution using gentle stirring, eluted at a shorter retention time of about 1.0 min. The increased concentration signal of the low molecular weight peak in the fractogram of glutenin dissolved using sonication (Fig. 1a) could result from the degraded high molecular weight material. Since the concentration signal of the high molecular weight fraction peak at 4 min was also higher when using

Fig. 1. (A and B): molecular weight distributions (W) across fractograms for glutenin dissolved by sonication (A: sample 1 in Table 1) and by gentle stirring (B: sample 6 in Table 1) with the 908 light scattering signal plotted with full line and the RI signal with dotted line. In average the weight average molecular weights, determined over the whole molecular weight distributions, were 5 £ 106 (A) and 2 £ 107 (B), respectively, which indicated partial degradation of the protein when using sonication. Molecular weights at the level of 1 £ 108 ; detected after using gentle stirring (B), could not be observed in solutions of glutenin dissolved using sonication (A). (a and b): characteristic fractograms from the RI signal from the FFF–MALS– RI analysis of glutenin dissolved by sonication (a: sample 1 in Table 1) shows a higher and more distinct low molecular weight fraction peak at 1.4 min (arrow) compared to the one obtained when using gentle stirring (b: sample 6 in Table 1) at 1 min (arrow). The increased sample mass dissolved by sonication compared to that by gentle stirring is shown by the increased high molecular weight peak height at about 4 min in (a) (arrow) compared to at about 3 min in (b) (arrow). AsFlFFF conditions: (A and a) Fin ¼ 5:2 ml/min, Fc ¼ 4:7 ml/min, Fout ¼ 0:5 ml/min, temperature ¼ 24 8C; (B and b) Fin ¼ 5:2 ml/min, Fc ¼ 4:2 ml/min, Fout ¼ 0:9 ml/min, temperature ¼ 24 8C.

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sonication, some of the increased concentration signal must be due to an increased total amount of material dissolved. This is due to the interrupted and repeated dissolutions used when glutenin was dissolved by gentle stirring. The total amount of sample material dissolved in the combined gentle stirring and the repeated stirring procedure was however almost in the same range as the amount dissolved by sonication. Since similar weight average molecular weights and molecular distributions were obtained for glutenin dissolved by both gentle stirring and repeated stirring it was concluded that the high molecular weight components were continuously dissolved and there were no preferential dissolution of glutenins with lower molecular weights. As a result the total amount of dissolved material could be almost as high in solutions of glutenin dissolved by gentle stirring as in those dissolved by sonication provided the solutions were stirred long enough. The highest molecular weight components of glutenin were, however, dissolved only at very low concentrations by gentle stirring. The complete absence of these components in the sonicated solutions suggests that they have been degraded into lower molecular weight species, thereby explaining the somewhat larger amount of total material dissolved and the increased concentration of low molecular weight material in these solutions. The molecular weights of the low molecular weight fractions were about the same in both sonicated and gently stirred samples, 5 £ 104 – 2 £ 105 (Fig. 1A). 3.2. Molecular weight distribution at different sonication powers MDs of the four differently sonicated preparations showed significant differences in the cumulative MDs as shown in Fig. 2 (Curves II, III, IV). The presence of different amounts of the low molecular weight fractions in the four sonicated samples caused the difference in MD but the maximum molecular weight was about the same as shown in

Fig. 2. Differences in molecular weight distribution between solutions of glutenin dissolved using sonication with different powers (II ¼ sample 1; III ¼ sample 2; IV ¼ sample 3) and using gentle stirring (I ¼ sample 6) as illustrated by the cumulative weight fraction.

Fig. 3 (Curves II, III, IV). The cumulative weight fraction also demonstrated the difficulty in dissolving the highest molecular weight components of glutenin using gentle stirring. As a result a lower amount of high molecular weight components were dissolved (Fig. 2, Curve I) but higher molecular weights could be observed (Fig. 3, Curve I) due to the very gentle dissolution method. The high molecular weight components, present in the solution of glutenin dissolved using gentle stirring, were obviously degraded when using sonication resulting in a lower Mw and a larger amount of low molecular weight components. 3.3. Rms radii The rms radii determined from the MALS –RI data was 225 ^ 25 nm for glutenin dissolved by gentle stirring. In accordance with the decrease in weight average molecular weight when using sonication the rms radius for all sonicated samples was also lower, (150 ^ 20) nm. As in the case of the determined Mw ; no significant difference in the rms radius between the differently sonicated samples could be noticed. The decrease in the measured rms radius further supports the suggested degradation of glutenin when using sonication. 3.4. Experimental artifacts A strong influence of certain parameters was observed on the retention time, most importantly the sample concentration, but perhaps also the orientation of the sample components in the separation channel and their conformation. Sample concentration has been noted previously to be a crucial parameter (Caldwell et al., 1988; Hansen et al., 1989; Litzen, 1992) especially when studying ultra-large macromolecules. Overloading effects

Fig. 3. The highest detectable molecular weight components in the molecular weight distributions of the sonicated glutenin samples were of about the same value in all samples (II ¼ sample 1; III ¼ sample 2; IV ¼ sample 3), but lower than those in the solutions of glutenin dissolved using gentle stirring (I ¼ sample 6). The overloading phenomena occurring in the FFF channel results in the fact that sample components of the same molecular weight are not being eluted from the channel at the same retention time. AsFlFFF conditions: II, III, IV Fin ¼ 5:2 ml/min, Fc ¼ 4:7 ml/min, Fout ¼ 0:5 ml/min, temperature ¼ 24 8C; I Fin ¼ 5:2 ml/min, Fc ¼ 4:2 ml/min, Fout ¼ 0:9 ml/min, temperature ¼ 24 8C.

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in FFF have often been identified as a shift in peak shape and position, as observed in this study. The result of this overloading is shown in Fig. 3 where sample components of the same molecular weight are being eluted at different retention times, due to differences in injected mass, even though the analytical conditions were the same. Erroneous information on the hydrodynamic size of the molecules thus arose from the calculations made from the observed retention times using FlFFF theory. The need for a high enough signal from both the MALS and the RI detector, in order to obtain accurate size determinations from these detectors, however made overloading necessary and unavoidable in some cases in this study. Since very large macromolecules were analysed, various factors contributing to the uncertainty of the MALS detection have to be taken into consideration. This concerns for example, Mie scattering, if the sample has a high density (Aberle et al., 1994), and a non-negligible A2 value (Wyatt, 1993). The use of injected sample concentrations , 2%, which are then diluted further in the AsFlFFF channel should however have kept the A2 term of the light scattering equation insignificant (van Dijk and Smit, 2000). Thus the reported molecular weights should be regarded as approximate.

4. Conclusions Using sonication to dissolve glutenin resulted in experimentally observed degradation of the protein. The observed weight average molecular weight and rms radius was lower compared with gentle stirring as the dissolution method. The MD in four differently sonicated samples also varied, which indicated that the degradation of glutenin depended upon the sonication power used. Thus sonication is concluded to be an efficient dissolution method. The sample can be dissolved in a couple of minutes compared several days for gentle stirring. However, this is at the cost of degradation into lower molecular weight materials. Sonication should therefore not be used if the largest, undegraded glutenin proteins are to be characterised. Dissolution by gentle stirring is then the only recommended method, accepting the longer dissolution times.

Acknowledgements This study was supported by the Swedish Council for Forestry and Agricultural Research, the Swedish Natural Science Research Council and the Crafoord Foundation. Mats Andersson at the Department of Technical Analytical Chemistry is acknowledged for discussions on multiangle light scattering measurements.

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References Aberle, T., Burchard, W., Vorwerg, W., Radosta, S., 1994. Conformational contributions of amylose and amylopectin to the structural-properties of starches from various sources. Starch –Starke 46, 329–335. Andersson, M., Wittgren, B., Wahlund, K.-G., 2001. Ultrahigh molar mass component detected in ethylhydroxyethyl cellulose by asymmetrical flow field-flow fractionation coupled to multiangle light scattering. Analytical Chemistry 73, 4852–4861. Astafieva, I.V., Eberlein, G.A., Wang, Y.J., 1996. Absolute on-line molecular mass analysis of basic fibroblast growth factors and its multimers be reversed-phase liquid chromatography with multi-angle laser light scattering detection. Journal of Chromatography, A 740, 215– 229. Bean, S.R., Lookhart, G.L., 2001. Factors influencing the characterization of gluten proteins by size-exclusion chromatography and multiangle laser light scattering (SEC–MALLS). Cereal Chemistry 78, 608–618. Berry, G.C., 1966. Thermodynamic and conformational properties of polystyrene. I. Light-scattering studies on diluted solutions of linear polystyrenes. Journal of Chemical Physics 44, 4550–4564. Bietz, J.A., 1984. Analysis of wheat gluten proteins by high-performance liquid chromatography. Baker’s Digest 58, 15– 17.(see also pages 20, 21 and 32). Bottomley, R.C., Kearns, H.F., Schofield, J.D., 1982. Characterisation of wheat flour and gluten proteins using buffers containing sodium dodecyl sulphate. Journal of the Science of Food and Agriculture 33, 481– 491. Caldwell, K.D., Brimhall, S.L., Gao, Y., Giddings, J.C., 1988. Sample overloading effects in polymer characterization by field-flow fractionation. Journal of Applied Polymer Science 36, 703 –719. Carceller, J.-L., Aussenac, T., 2001. Size characterization of glutenin polymers by HPSEC–MALLS. Journal of Cereal Science 33, 131–142. Danno, G., 1981. Extraction of unreduced glutenin from wheat flour with sodium dodecyl sulfate. Cereal Chemistry 58, 311–313. Danno, G., Hoseney, R.C., 1982. Changes in flour proteins during dough mixing. Cereal Chemistry 59, 249 –253. Danno, G., Kanazawa, K., Natake, M., 1974. Extraction of wheat flour proteins with sodium dodecyl sulfate and their molecular weight distribution. Agricultural and Biological Chemistry 38, 1947–1953. van Dijk, J.A.A.P., Smit, J.A.M., 2000. Size-exclusion chromatography – multiangle laser light scattering analysis of B-lactoglobulin and bovine serum albumin in aqueous solution with added salt. Journal of Chromatography, A 867, 105–112. Eisenberg, H., 1976. Partial volume, density and refractive-index increments. Biological macromolecules and polyelectrolytes in solution, Clarendon Press, Oxford, pp. 48– 63. Eliasson, A.-C., 1993. Cereals in breadmaking: a molecular colloidal approach. In: Eliasson, A.-C., Larsson, K. (Eds.), Cereals in Breadmaking, Marcel Dekker, New York, pp. 57– 69. Folta-Stogniew, E., Williams, K.R., 1999. Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. Journal of Biomolecular Techniques 10, 51–63. Giddings, J.C., 1993. Field-flow fractionation: analysis of macromolecular, colloids, and particulate materials. Science 260, 1456–1465. Giddings, J.C., Yang, F.J., Myers, M.N., 1977. Flow field-flow fractionation as a methodology for protein separation and characterization. Analytical Biochemistry 81, 395–407. Giddings, J.C., Benincasa, M.-A., Liu, M.-K., Li, P., 1992. Separation of water soluble synthetic and biological macromolecules by flow field-flow fractionation. Journal of Liquid Chromatography 15, 1729–1747. Hansen, M.E., Giddings, J.C., Beckett, R., 1989. Colloid characterization by sedimentation field-flow fractionation VI. Perturbations due to

8

C. Arfvidsson et al. / Journal of Cereal Science 39 (2004) 1–8

overloading and electrostatic repulsion. Journal of Colloid and Interface Science 132, 300–312. Jumel, K., Fiebrig, I., Harding, S.E., 1996. Rapid size distribution and purity analysis of gastric mucus glycoproteins by size exclusion chromatography/multi angle light scattering. International Journal of Biological Macromolecules 18, 133 –139. Kameyama, K., Nakae, T., Takagi, T., 1982. Estimation of molecular weights of membrane proteins in the presence of SDS by low-angle laser light scattering combined with high-performance porous silica gel chromatography. Biochimica Biophysica Acta 706, 19–26. Litze´n, A., 1992. Asymmetrical flow FFF. Doctoral dissertation. University of Uppsala Litze´n, A., 1993. Separation speed, retention and dispersion in asymmetrical flow field-flow fractionation as functions of channel dimensions and flow rates. Analytical Chemistry 65, 461–470. Litze´n, A., Walter, J.K., Krischollek, H., Wahlund, K.-G., 1993. Separation and quantitation of monoclonal antibody aggregates by asymmetrical flow field-flow fractionation and comparison to gel permeation chromatography. Analytical Biochemistry 212, 469 –480. Lookhart, G.L., 1997. New methods helping to solve the glutenin puzzle. Cereal Foods World 42, 16–19. Lundh, G., MacRitchie, F., 1989. Size exclusion HPLC characterisation of gluten protein fractions varying in breadmaking potential. Journal of Cereal Science 10, 247–253. MacRitchie, F., 1975. Mechanical degradation of gluten proteins during high-speed mixing of doughs. Journal of Polymer Science, Polymer Symposia 49, 85–90. MacRitchie, F., 1987. Evaluation of contributions from wheat protein fractions to dough mixing and breadmaking. Journal of Cereal Science 6, 259 –268. MacRitchie, F., 1992. Physicochemical properties of wheat proteins in relation to functionality. Advances in Food Nutrition Research 36, 1– 87. Miyake, J., Takagi, T., 1981. A low angle laser light scattering study of the association behavior of the major membrane protein of Rhodospirillum rubrum chromatophore at various concentrations of sodium dodecyl sulfate where polypeptides derived from water-soluble globular proteins are solubilized monomerically. Biochimica Biophysica Acta 668, 290–298. Moonen, J.H.E., Scheepstra, A., Graveland, A., 1982. Use of the SDSsedimentation test and SDS–polyacrylamide gel electrophoresis for screening breeder’s samples of wheat for bread making quality. Euphytica 31, 677–690. Morel, M.-H., Dehlon, P., Autran, J.C., Leygue, J.P., Bar-L’Helgouac´h, C., 2000. Effects of temperature, sonication time and power settings on size distribution and extractability of total wheat flour proteins as determined by size-exclusion high performance liquid chromatography. Cereal Chemistry 77, 685 –691. Myers, M.N., 1997. Overview of field-flow fractionation. Journal of Microcolumn Separations 9, 151–162. Reynolds, J.A., Tanford, C., 1970. Binding of dodecyl sulfate to proteins at high binding ratios. Possible implications for the state of proteins in

biological membranes. Proceedings of the National Academy of Science USA 66, 1002– 1007. Singh, N.K., MacRitchie, F., 1989. Controlled degradation as a tool for probing wheat protein structure. In: Salovaara, H., (Ed.), Wheat end-use properties. Wheat and flour characterization for specific end uses, University of Helsinki, Helsinki, pp. 321 –326. Singh, N.K., Donovan, G.R., Batey, I.L., MacRitchie, F., 1990. Use of sonication and size-exclusion high-performance liquid chromatography in the study of wheat flour proteins. I. Dissolution of total proteins in the absence of reducing agents. Cereal Chemistry 67, 150 –161. Southan, M., MacRitchie, F., 1999. Molecular weight distribution of wheat proteins. Cereal Chemistry 76, 827–836. Stevenson, S.G., Preston, K.R., 1996. Flow field-flow fractionation of wheat proteins. Journal of Cereal Science 23, 121 –131. Stevenson, S.G., Preston, K.R., 1997. Effects of surfactants on wheat protein fractionation by flow field-flow fractionation. Journal of Liquid Chromatography and Related Technologies 20, 2835–2842. Takagi, T., Miyake, J., Nashima, T., 1980. Assessment study on the use of the low angle laser light scattering technique for the estimation of molecular weight of the polypeptide forming a complex with sodium dodecyl sulfate. Biochimica Biophysica Acta 626, 5– 14. Thielking, H., Kulicke, W.M., 1996. On-line coupling of flow field-flow fractionation and multiangle laser light scattering for the characterization of macromolecules in aqueous solution as illustrated by sulfonated polystyrene samples. Analytical Chemistry 68, 1169–1173. Thielking, H., Roessner, D., Kulicke, W.M., 1995. Online coupling of flow field-flow fractionation and multiangle laser-light scattering for the characterization of polystyrene particles. Analytical Chemistry 67, 3229–3233. Wahlund, K.-G., Giddings, J.C., 1987. Properties of an asymmetrical flow field-flow fractionation channel having one permeable wall. Analytical Chemistry 59, 1332–1339. Wahlund, K.-G., Gustavsson, M., MacRitchie, F., Nylander, T., Wannerberger, L., 1996. Size characterisation of wheat proteins, particularly glutenin, by asymmetrical flow field-flow fractionation. Journal of Cereal Science 23, 113– 119. Weegels, P.L., Flissebaalje, T., Hamer, R.J., 1994. Factors affecting the extractability of the glutenin macropolymer. Cereal Chemistry 71, 308 –309. Wen, J., Arakawa, T., Philo, J.S., 1996. Size-exclusion chromatography with on-line light-scattering, absorbance, and refractive index detectors for studying proteins and their interactions. Analytical Biochemistry 240, 155–166. Wittgren, B., Wahlund, K.-G., 1997. Fast molecular mass and size characterization of polysaccharides using asymmetrical flow field-flow fractionation–multiangle light scattering. Journal of Chromatography, A 760, 205–218. Wyatt, P.J., 1993. Light scattering and the absolute characterization of macromolecules. Analytical Chimica Acta 272, 1–40.