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Conformational studies of wheat gluten, glutenin, and gliadin in urea solutions at various pH's
ARCHIVES
OF
BIOCHEMISTRY
Conformational
AND
BIOPHYSICS
435-410
107,
Studies
of Wheat
in Urea
Solutions
T. VICTOR From the Northern
(1964)
Gluten, at Various
WI? AIVD ROBERT Regional Received
Glutenin,
Gliadin
pH’s
J. DIMLER
Research Laborator?g,l March
and
Peoria,
Illinois
30, 1964
Viscosity, sedimentation velocity, ultraviolet difference spectra, and optical rotatory dispersion measurements were carried out on wheat gluten, glutenin, and gliadin in 3 111urea plus 0.11 fif KC1 plus 0.02 2cZbuffer at pH 3-10 at 25°C or room temperature. An increase in intrinsic viscosity and a decrease in sedimentation coefficient for glutenin at pH 10 compared with that at pH 4 are consistent with an increase in asymmetry of the protein molecule. Parameters from optical rotatory dispersion studies on glutenin also indicate a conformational change at pH 10. Some increases in intrinsic viscosity were also observed for gluten and gliadin at pH 10, but the increase for gliadin might not be significant. The absence of tyrosine and tryptophan peaks in the ultraviolet difference spectra of gluten, glutenin, and gliadin suggests t,hat these two amino acids are not involved in any interaction with other groups.
Wheat gluten cau be separated into 70% ethanol-soluble gliadin and 70 5% ethanolinsoluble glutenin by the classical method of Osborne (1). Starch-gel electrophoresis with an aluminum lactate buffer (pH 3.1) demonstrated that the high molecular weight fraction, glutenin, was unable to migrate in starch gels and that the gliadin fraction was composed of at least eight components (2). The amino acid compositions of the gliadin components and fractions separated by column chromatography are very similar (3) and not greatly different from the composition of glutenin. Hydrogen-ion equilibria of gluten (4), glutenin, and gliadin (5) show that the empirical electrostatic factor w at acid pH was considerably larger than it was at alkaline pH, and this difference suggested other physicochemical studies in the same titrating solvent, 3 M urea plus 0.15 111KCI. This paper reports the viscosity, sedirnentation velocity, difference spectra, and optical 1 This is a laboratory of t,he Northern I!tilization Research and L)evelopment Division, Agricultural Research Service, IJ. S. Ilepartment, of Agriculture.
rotatory gliadin.
dispersion of gluten, glutenin, MATERIALS
AND
and
METHODS
The gluten, glutenin, and gliadin from Ponca hard red winter wheat were from the same preparation, according to the procedure of Jones et al. (Gj, t,hat was used in determining hydrogen-ion equilibria (4, 5). The concentration of each protein solution was determined at 276 mp in a Cary 11 recording spectrophotometer (Applied Physics Corporation”), and the same conversion factor was used (4, 5). Reagent grade chemicals and deionized distilled water were used throughout. \?scosity measurements were made at four protein concentrations (O.l-1.3\() with CannonFenske No. 50 viscometers and a flow time of about 250 seconds for wat,er. The viscometers were in a constant, t,emperature water bath at 25” & O.Ol”C. Intrinsic viscosity was obtained t)y extrapolating the plots of (q-l)/c vs. c and (In ~,)/c vs. c to zero concentration. Sedimentation velocity measurements at room temperature (22”2S”C) were made with a Rpinco model IS analytical ultracentrifuge with an RTIC unit for temperature regulation within 0.1”. A ~-~~ 2 Mention of firm names or trade product,s is for identification only and does not imply endorsement by the IT. S. I>epartment of Agriculture. 435
436
WU AXD
carbon-filled epoxy centerpiece and a 30-mm cell were used at 47,660 rpm for gluten and gliadin and at 31,410 rpm for glutenin. The sedimentation coefficient, s, was calculated according to the equation (7): 2(X2 - 2,) s = (22 + z&At* - t,) ’
DIMLER For glutenin the uncertainty was about twice these values due to the lower concentration used. The pH’s were measured with a Radiometer pH meter 4 calibrated with standard phthalate and phosphate buffers prepared as described by Bates (8).
(1)
where w is the angular velocity in radians per second, t is the time in seconds, and z is the distance of the boundary in centimeters from the axis of rotation. Values of s were calculated when t is 8460 or 9420 seconds. All measurements were made after enlarged patterns had been traced on paper. Difference spectra were also measured in the Cary spectrophotometer equipped with l-cm glass-stoppered silica cells. Both cells contained protein at the same concentration. Water flowing t,hrough a, specially constructed block in which the measuring cell was located kept the temperature of the solution in its compartment constant within the selected range from 25” to 90’. The temperature in the reference compartment was kept at room temperature. The difference spectra were recorded from 350 to about 241 rnb, where the slit width became maximum. Whenever a 10” change was made, 10 minutes proved adequate to allow for thermal eyuilibrium after the bath had reached the desired temperature, which was constant within 0.1” at lower levels and within 0.15” at 90”. Measurements of optical rotatory dispersion were made at room temperature with a Bellingham & Stanley polarimeter modified for photoelectric reading by 0. C. Rudolph & Sons. A Bausch di Lomb grating monochromator with a mercury arc source was used. The solutions to be measured were contained in a water-jacketed locm quartz polarimeter tube of 7-mm bore with fused quartz end-plates made by American Instrument Co. The indices of refraction, 12, of the solvents used were determined with a Bausch & Lomb refractometer at 25”. The optical rotation was measured at 579, 546, 436,405, 365, and 313 rnp. At the end of each set of measurements it was measured again at 579 ml; no change in optical rotation was noticed. Solvent blanks were handled similarly, and the blank was subtracted from the observed rotation of the protein solution. The specific rotation [01] was then calculated. The protein concentration was l”/; from 579 to 365 rnr and 0.2% for 313 rnp. The optical rotation of a 0.2% protein solution was also measured at 405 and 365 mM, and the specific rotation was independent of the protein concentration in the range studied. The degree of precision of [a] was about &3” at 579 mp and &8” at 313 ,110.
RESULTS
AND
DISCUSSION
The intrinsic viscosities [q] of gluten, glutenin, and gliadin in 3 ill’ urea plus 0.11 M KC1 plus 0.02 ill buffer are shown in Table I. For pH 3 and Yj gluten solutions, viscosity was independent of time. A decrease in viscosity with time was observed for pH 10 solution. In Table I the viscosity of pH 10 solution was extrapolated to zero time. A relatively large increase in intrinsic viscosity at high pH was seen for glutenin. Smaller increases were observed for gluten and gliadin. The viscosity change for whole gluten is greater than that for gliadin because the glutenin component strongly influences the viscosity of whole gluten. The increase in intrinsic viscosity indicates an increase of asymmetry or effective volume of the molecule. The sedimentation coefficients of gluten, glutenin, and gliadin at various pH’s are shown in Table II. Since the experiments were at different temperatures, the observed s values calculated from Eq. (1) were corrected by a factor ~~T/v~ocorresponding to the viscosity of water at T” relative to that at 20°C. The ~~0,~ values were not calculated because only the relative values of s were of concern here. The last gluten sample in Table II was prepared and used considerably sooner than the other samples, and no differTABLE INTRINSIC
I
VISCOSITY OF GLUTEN, GLIADIN IN 3 M UREA BUFFER AT 25°C
Wheat Protein
Gluten
Glutenin Gliadin
a Extrapolated
GLUTENIN, PLUS
AND
PH
[rll (100ml/sm)
3.3-3.5 5.4 10.1-10.2 4.1-4.3 10.3 4.13.3 10.0-10.2
0.276 0.264 0.310 0.58 0.82a 0.18 0.19”
to zero time.
CONFORMATIONAL TABLE
STUDIES
OF WHErlT
II
GLUTES,
GLUTENIN
AND
GLIBDIN
$j$
solution had a pH of 7.97 after cooling to roonl tenlperature. The change in pH is probably a result of deconlposition of sonle urea at high tenlperature. Another solution originally of pH 8 had 8.56 after heating sch.~~~;/112uL M’heat Protein Cont. (7, PH to 90” and cooling to roonl tenlperature and gave a difference spectrunl sinlilar to Fig. 2. Gluten 0.18 3.3 1.40 Other solutions of gluten in buffers of vary0.95 1.N 3.4 ing pH’s front 2 to 1l.Z were between 7.5 0.54 5.3 1.39 and 9 after heating to 90” and cooling. The 10.2 0.50 1.42 0.50 10.2 1.41 tyrosyl groups in gluten are norinal (1), (Xiadin 0.33 4.1 1.46 and the heat of ionization of a normal 10.2 0.33 1.44 tyrosyl group is 6000 cal per mole (11). The Glutenin 0.34 4.3 4.28 use of the van% Hoff equation d ln K,‘dT = 10.4 0.35 3.2G AH,/RTz gave a pK value of 9.5 for tyrosine at 90” as conlpared to 10.3 at 25” (1). The ence in s value was observed. No time de- snlall positive peak at 29,5 111~in Fig. 2 is pendence of s and no concentration de- likely due to enhanced tyrosyl ionization pendence of s were observed. Since an at 90”. At low and near neutral pH, the peaks in increase of asymmetry of a molecule will give a lower sedimentation coefficient (9), a sig- the ultraviolet difference spectra of prot#eins below 275 111~usually arise front perturbanificant decrease in sedimentation coefficient for glutenin at high pH is consistent with an tions of phenylalanyl and histidyl chromoincrease of asynunetry of the glutenin mole- phores, those between 275 and 290 nip frorn tyrosyl chronlophore, and that above 290 111~ cule. Essentially the same sedimentation chronlophore (12, 13). coefficient for gluten and gliadin was ob- front tryptophanyl The absence of difference spectra front pH 3 tained because glutenin had already nligrated off the peak in gluten solution before to 8 at room tenlperature and of characteristic peaks for tyrosyl and tryptophanyl au accurate measurement could be taken. chronlophores at 90” suggests that no aroDifference spectra of each protein at various pH’s were measured against the same nlatic group is involved at roonl tenlperature protein solution of pH 7.09 as reference. and that these two groups are likely not When a solution at pH 3.57, .5.62, or 8.00 involved at 9Oo.3When tyrosyl group ionizes was used in the proper compartment at room at alkaline pH, then the peaks at 29.5 and 245 nip appear. The spectruln below 27;i 111~ temperature (23 =t a’), there was essentially no observable spectrum in the region 240- in Fig. 2 indicates the possible involvenlent $50 mp. Two peaks at 29.5 and 245 nip be- of phenylalanyl and histidyl groups at high tenlperature. Since little is known about the came evident as pH of the protein solution perturbation of phenylalanyl and histidyl increased to 9.16. Figure 1 is a typical differgroups and since the possible decoluposition ence spectrum in alkaline pH where the magnitudes of 295 and 24.5 nip peaks in- of sonle urea at high tenlperature further no positive crease with increasing pH in the region of conlplicates the interpretation, tyrosyl ionization. The shape of I:ig. 1 and statenlent seenls justified at this tinle. It the ratio of the maxima at 245 and 295 111~ lnay be noted, however, that the difference are very close to the difference spectra of spectra of gluten, glutenin, and gliadin are tyrosiue and heme proteins reported by quitBe different from that of soybean trypsin Hermans (10) when a solution at high pH 3The tyrosyl groups ionixe normally (4). Alwas read against a solution of the same corn though the possibility remains that tyrosyl and ccntration at neutral pH. Figure 2 is a tryptophanyl groups might be involved in some typical difference spectrum at high temperainteractions not influenced by change of pH and ture. Roth solutions in Fig. 2 had the sanle by heating to 90’ such a possibility is not, very starting pH of 7.09. However the heated likely to exist. SEDIMENTATION COEFFICIENT or GLCTEN, GLUTENIN, AND GLIADIN IN 3 M VREA PLUS Bc-FPER AT ROOM TEMPERATCRE
438
WU AND
DIMLER
Wavelength, mp FIG. 1. Difference spectra of gluten at 25” in 3 ik! urea plus 0.11 IM KC1 plus 0.02 M buffer; pH 9.63 vs. pH 7.09 as reference. The optical density of the reference was set at zero at 350 mr. Gluten concentration was 2.37 mg. per milliliter in each solution. I
I
I
I
I
I
I
I
I
If
0.5 -
!
x Z .Y Z 0 0 I
k!b 250
I
300 Wavelength, mp
I
I
I
I
350
FIG. 2. Difference spectra of gluten at pH 7.97, 90.0”, in 3 M urea plus 0.11 M KC1 plus 0.02 M buffer vs. gluten at pH 7.09 (reference set at zero optical density at 350 mp), 30.5” in the same solvent. Gluten concentration 2.37 mg. per milliliter in both solutions.
inhibitor (12), where large tyrosyl and tryptophanyl peaks are evident at high temperature. Optical rotatory dispersion curves for gluten, glutenin, and gliadin were plotted by two methods. The first method was according to nloffitt (14):
value of X0 was taken to be 212 m/~ in accordance with that found best for poly--benzyl-L-glutamate in a variety of solvents (15). A typical Moffitt plot is shown in Fig. 3. The parameter bo was calculated from (Ml 100)[3/(n2 + 2)]/x04 times the slope of a plot of [a] (X2 - X,2) vs. l/(Xf - X,2). Since bo in Eq. (2) was small, the data were also plotted according to Drude equation (16): X2[a] = Xc$] + k.
where M is the average residue weight and n is the refractive index of the solvent. The
(3)
A Drude plot for gluten is shown in Fig. 4. The parameter X, was obtained from the slope of the straight line of X2[01]vs. [a]. A
CONFORMATIONAL
STUDIES
OF WHEAT
GLUTEN,
GLUTENIN
AND GLIADIN
-239
? = -30 x “do -32 I “2 -340 z?J
I 0 0-0, I 5
I
I
I 15
I 20
I @y-Q I 10
25
1
x 106 A= - A',
FIG. 3. A Moffitt plot of the rotatory dispersion of gluten in 3 111urea plus 0.09 M KC1 plus 0.04 llri borate at. room temperature, pH 10.1. Gluten concentrations 0.99 and 0.2252.
FIG. 4. A Drude plot of the rotatory dispersion of gluten in 3 M urea plus 0.09 M KC1 plus 0.04 iM borate at room temperature, pH 10.1. Gluten concentrations 0.99 and 0.22yc.
sumnary of the optical rotation data is given in Table III. Theoretically, the value for gluten should be a weighted average of glutenin and gliadin. However, the limited accuracv of the oolarimeter and the low solubility of gluteiin caused some apparent inconsistency of the gluten value from that of the average of glutenin and gliadin. The values in Table III, however, are accurate enough to make a qualitative statement and a relative comparison. The mall difference in values of bo , a0 , X, , and [CY]Wfor gluten and gliadin at high and low pH’s is within experimental error and considered not significant. There seems, however, a significant change in the optical rotatory parameters of glutenin between high and low pH. The same change in bo values was observed for glutenin in 8 M urea solution (17). If the gluten proteins can be approximated as a mixture of random coil and helix, then low
TABLE
III
OPTICAL ROTATORY PROPERTIES OF GLUTEN, GLUTENIN, AND GLIADIN IN 3 M UREA PLIX BUFFER AT ROOM TEMPERATCRE Wheat protein
Glut,en Gliadin
’ pH I 10.1 3.8 10.2
-40
4.4
Glutenin
10.3 4.2
-
-50
-622 -642
-108 -90 -52 -11
-688 -721 -596 -673
217 220 225 224 222 213
helical contents (perhaps below 20 Lil) are exhibited by all three proteins with gliadin having the highest value. The change in bo for glutenin at high pH is in the directiou of increasing helical content. Kretschmer (18) interpreted his optical rotatory dispersion measureulents according
440
WU AND
to Moffitt’s theory (15) and indicated that wheat gluten in 0.01 M formic acid and gliadin in 70 % ethanol have helical contents of about 35 %. Kretschmer’s results cannot be compared with ours because of the different solvents used. Glutenin has five disulfide groups per lo5 gm from amino acid analysis (5) ; disulfide-cleaved glutenin has a molecular weight of 20,000 and a much smaller intrinsic viscosity (19). An increase of intrinsic viscosity of glutenin was around pH 10 (Table I) ; this increase indicates that no disulfide bond is cleaved at least in the initial period when measurements were made. Glutenin showed a very large decrease in empirical electrostatic factor w at high pH from hydrogen in equilibria (5). Since w depends on molecular size, shape, and permeability at constant temperature and ionic strength, a decrease in w can result from an increase in molecular size, asymmetry, or permeability. For glutenin, at least, an increase in asymmetry of the molecule at high pH is supported by the following evidence, which includes: (1) An increase in intrinsic viscosity, (2) a decrease in sedimentation coefficient, and (3) a change in optical rotatory parameters in the direction of increasing helical content. Taylor and Cluskey (20) measured the intrinsic viscosities of glutenin and gliadin in sodium lactate buffer of pH 3.1 at ionic strengths 0.003 and 0.03. They concluded that glutenin behaves as a flexible, randomly coiled polyelectrolyte but that gliadin has a low, relatively constant, intrinsic viscosity, which perhaps indicates a comparatively globular protein. For gliadin, local electrostatic effect or an increase in permeability may cause a relatively small decrease in w at high pH from hydrogen-ion equilibria (5). Gliadin only shows small changes in viscosity, sedimentation coeficient, and optical rotatory parameters, and these changes border on experimental un-
DIMLER
certainties. Further physicochemical studies on glutenin and gliadin are planned to supplement the data available at present. ACKNOWLEDGMENT We thank sedimentation
G. E. Babcock for carrying velocity runs.
out the
REFERENCES 1. OSBORNE, T. B., “The Vegetable Proteins,” 2nd edition. Longmans, Green, New York, 1924. 2. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., Arch. Biochem. Biophys. 94,477 (1961). 3. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., J. Agr. Food Chem. 9, 307 (1961). 4. Wu, Y. V., AND DIMLER, R. J., Arch. Biochem. Biophys. 102, 230 (1963). 5. Wu, Y. V., AND DIMLER, R. J., Arch. Biochem. Biophys. 103, 310 (1963). 6. JONES, R. W., TAYLOR, N. W., AND SENTI, F. R., Arch. Biochem. Biophys. 84,363 (1959). in 7. SCHACHMAN, H. K., “Ultracentrifugation Biochemistry.” Academic Press, New York, 1959. pH Determina8. BATES, R. G., “Electrometric tions.” Wiley, New York, 1954. Chem. 16, 341 9. YANG, J. T., Advan. Protein (1961). 1, 193 (1962). 10. HERMANS, JAN, JR., Biochemistry 11. TANFORD, C., SWANSON, S. A., AND SHORE, W. S., J. Am. Chem. Sot. 77, 6414 (1955). 12. Wu, Y. T’., AND SCHERAGA, H. A., Biochemistry 1, 995 (1962). A., “Protein Structure.” 13. SCHERAGA, H. Academic Press, New York, 1961. 14. MOFFITT, W., J. Chew. Phys. 25, 467 (1956). 15. MOFFITT, W., AND YANG, J. T., Proc. Nal.!. Acad. Sci. U.S. 49, 596 (1956). 16. YANG, J. T., AND DOTY, P. M., J. Anl. Chem. Sot. 79, 761 (1957). 17. ZOBEL, H. F., AND TAYLOR, N. W., In preparation. 18. KRETSCHMER, C. B., J. Phys. Chem. 61, 1627 (1957). 19. NIELSEN, H. C., BABCOCK, G. E., AND SENTI, F. R., Arch. Biochem. Biophys. 96,252 (1962). 20. TAYLOR, N. W., AND CLUSKEY, J. E., Arch. Biochem. Biophys. 97, 399 (1962).
OF
BIOCHEMISTRY
Conformational
AND
BIOPHYSICS
435-410
107,
Studies
of Wheat
in Urea
Solutions
T. VICTOR From the Northern
(1964)
Gluten, at Various
WI? AIVD ROBERT Regional Received
Glutenin,
Gliadin
pH’s
J. DIMLER
Research Laborator?g,l March
and
Peoria,
Illinois
30, 1964
Viscosity, sedimentation velocity, ultraviolet difference spectra, and optical rotatory dispersion measurements were carried out on wheat gluten, glutenin, and gliadin in 3 111urea plus 0.11 fif KC1 plus 0.02 2cZbuffer at pH 3-10 at 25°C or room temperature. An increase in intrinsic viscosity and a decrease in sedimentation coefficient for glutenin at pH 10 compared with that at pH 4 are consistent with an increase in asymmetry of the protein molecule. Parameters from optical rotatory dispersion studies on glutenin also indicate a conformational change at pH 10. Some increases in intrinsic viscosity were also observed for gluten and gliadin at pH 10, but the increase for gliadin might not be significant. The absence of tyrosine and tryptophan peaks in the ultraviolet difference spectra of gluten, glutenin, and gliadin suggests t,hat these two amino acids are not involved in any interaction with other groups.
Wheat gluten cau be separated into 70% ethanol-soluble gliadin and 70 5% ethanolinsoluble glutenin by the classical method of Osborne (1). Starch-gel electrophoresis with an aluminum lactate buffer (pH 3.1) demonstrated that the high molecular weight fraction, glutenin, was unable to migrate in starch gels and that the gliadin fraction was composed of at least eight components (2). The amino acid compositions of the gliadin components and fractions separated by column chromatography are very similar (3) and not greatly different from the composition of glutenin. Hydrogen-ion equilibria of gluten (4), glutenin, and gliadin (5) show that the empirical electrostatic factor w at acid pH was considerably larger than it was at alkaline pH, and this difference suggested other physicochemical studies in the same titrating solvent, 3 M urea plus 0.15 111KCI. This paper reports the viscosity, sedirnentation velocity, difference spectra, and optical 1 This is a laboratory of t,he Northern I!tilization Research and L)evelopment Division, Agricultural Research Service, IJ. S. Ilepartment, of Agriculture.
rotatory gliadin.
dispersion of gluten, glutenin, MATERIALS
AND
and
METHODS
The gluten, glutenin, and gliadin from Ponca hard red winter wheat were from the same preparation, according to the procedure of Jones et al. (Gj, t,hat was used in determining hydrogen-ion equilibria (4, 5). The concentration of each protein solution was determined at 276 mp in a Cary 11 recording spectrophotometer (Applied Physics Corporation”), and the same conversion factor was used (4, 5). Reagent grade chemicals and deionized distilled water were used throughout. \?scosity measurements were made at four protein concentrations (O.l-1.3\() with CannonFenske No. 50 viscometers and a flow time of about 250 seconds for wat,er. The viscometers were in a constant, t,emperature water bath at 25” & O.Ol”C. Intrinsic viscosity was obtained t)y extrapolating the plots of (q-l)/c vs. c and (In ~,)/c vs. c to zero concentration. Sedimentation velocity measurements at room temperature (22”2S”C) were made with a Rpinco model IS analytical ultracentrifuge with an RTIC unit for temperature regulation within 0.1”. A ~-~~ 2 Mention of firm names or trade product,s is for identification only and does not imply endorsement by the IT. S. I>epartment of Agriculture. 435
436
WU AXD
carbon-filled epoxy centerpiece and a 30-mm cell were used at 47,660 rpm for gluten and gliadin and at 31,410 rpm for glutenin. The sedimentation coefficient, s, was calculated according to the equation (7): 2(X2 - 2,) s = (22 + z&At* - t,) ’
DIMLER For glutenin the uncertainty was about twice these values due to the lower concentration used. The pH’s were measured with a Radiometer pH meter 4 calibrated with standard phthalate and phosphate buffers prepared as described by Bates (8).
(1)
where w is the angular velocity in radians per second, t is the time in seconds, and z is the distance of the boundary in centimeters from the axis of rotation. Values of s were calculated when t is 8460 or 9420 seconds. All measurements were made after enlarged patterns had been traced on paper. Difference spectra were also measured in the Cary spectrophotometer equipped with l-cm glass-stoppered silica cells. Both cells contained protein at the same concentration. Water flowing t,hrough a, specially constructed block in which the measuring cell was located kept the temperature of the solution in its compartment constant within the selected range from 25” to 90’. The temperature in the reference compartment was kept at room temperature. The difference spectra were recorded from 350 to about 241 rnb, where the slit width became maximum. Whenever a 10” change was made, 10 minutes proved adequate to allow for thermal eyuilibrium after the bath had reached the desired temperature, which was constant within 0.1” at lower levels and within 0.15” at 90”. Measurements of optical rotatory dispersion were made at room temperature with a Bellingham & Stanley polarimeter modified for photoelectric reading by 0. C. Rudolph & Sons. A Bausch di Lomb grating monochromator with a mercury arc source was used. The solutions to be measured were contained in a water-jacketed locm quartz polarimeter tube of 7-mm bore with fused quartz end-plates made by American Instrument Co. The indices of refraction, 12, of the solvents used were determined with a Bausch & Lomb refractometer at 25”. The optical rotation was measured at 579, 546, 436,405, 365, and 313 rnp. At the end of each set of measurements it was measured again at 579 ml; no change in optical rotation was noticed. Solvent blanks were handled similarly, and the blank was subtracted from the observed rotation of the protein solution. The specific rotation [01] was then calculated. The protein concentration was l”/; from 579 to 365 rnr and 0.2% for 313 rnp. The optical rotation of a 0.2% protein solution was also measured at 405 and 365 mM, and the specific rotation was independent of the protein concentration in the range studied. The degree of precision of [a] was about &3” at 579 mp and &8” at 313 ,110.
RESULTS
AND
DISCUSSION
The intrinsic viscosities [q] of gluten, glutenin, and gliadin in 3 ill’ urea plus 0.11 M KC1 plus 0.02 ill buffer are shown in Table I. For pH 3 and Yj gluten solutions, viscosity was independent of time. A decrease in viscosity with time was observed for pH 10 solution. In Table I the viscosity of pH 10 solution was extrapolated to zero time. A relatively large increase in intrinsic viscosity at high pH was seen for glutenin. Smaller increases were observed for gluten and gliadin. The viscosity change for whole gluten is greater than that for gliadin because the glutenin component strongly influences the viscosity of whole gluten. The increase in intrinsic viscosity indicates an increase of asymmetry or effective volume of the molecule. The sedimentation coefficients of gluten, glutenin, and gliadin at various pH’s are shown in Table II. Since the experiments were at different temperatures, the observed s values calculated from Eq. (1) were corrected by a factor ~~T/v~ocorresponding to the viscosity of water at T” relative to that at 20°C. The ~~0,~ values were not calculated because only the relative values of s were of concern here. The last gluten sample in Table II was prepared and used considerably sooner than the other samples, and no differTABLE INTRINSIC
I
VISCOSITY OF GLUTEN, GLIADIN IN 3 M UREA BUFFER AT 25°C
Wheat Protein
Gluten
Glutenin Gliadin
a Extrapolated
GLUTENIN, PLUS
AND
PH
[rll (100ml/sm)
3.3-3.5 5.4 10.1-10.2 4.1-4.3 10.3 4.13.3 10.0-10.2
0.276 0.264 0.310 0.58 0.82a 0.18 0.19”
to zero time.
CONFORMATIONAL TABLE
STUDIES
OF WHErlT
II
GLUTES,
GLUTENIN
AND
GLIBDIN
$j$
solution had a pH of 7.97 after cooling to roonl tenlperature. The change in pH is probably a result of deconlposition of sonle urea at high tenlperature. Another solution originally of pH 8 had 8.56 after heating sch.~~~;/112uL M’heat Protein Cont. (7, PH to 90” and cooling to roonl tenlperature and gave a difference spectrunl sinlilar to Fig. 2. Gluten 0.18 3.3 1.40 Other solutions of gluten in buffers of vary0.95 1.N 3.4 ing pH’s front 2 to 1l.Z were between 7.5 0.54 5.3 1.39 and 9 after heating to 90” and cooling. The 10.2 0.50 1.42 0.50 10.2 1.41 tyrosyl groups in gluten are norinal (1), (Xiadin 0.33 4.1 1.46 and the heat of ionization of a normal 10.2 0.33 1.44 tyrosyl group is 6000 cal per mole (11). The Glutenin 0.34 4.3 4.28 use of the van% Hoff equation d ln K,‘dT = 10.4 0.35 3.2G AH,/RTz gave a pK value of 9.5 for tyrosine at 90” as conlpared to 10.3 at 25” (1). The ence in s value was observed. No time de- snlall positive peak at 29,5 111~in Fig. 2 is pendence of s and no concentration de- likely due to enhanced tyrosyl ionization pendence of s were observed. Since an at 90”. At low and near neutral pH, the peaks in increase of asymmetry of a molecule will give a lower sedimentation coefficient (9), a sig- the ultraviolet difference spectra of prot#eins below 275 111~usually arise front perturbanificant decrease in sedimentation coefficient for glutenin at high pH is consistent with an tions of phenylalanyl and histidyl chromoincrease of asynunetry of the glutenin mole- phores, those between 275 and 290 nip frorn tyrosyl chronlophore, and that above 290 111~ cule. Essentially the same sedimentation chronlophore (12, 13). coefficient for gluten and gliadin was ob- front tryptophanyl The absence of difference spectra front pH 3 tained because glutenin had already nligrated off the peak in gluten solution before to 8 at room tenlperature and of characteristic peaks for tyrosyl and tryptophanyl au accurate measurement could be taken. chronlophores at 90” suggests that no aroDifference spectra of each protein at various pH’s were measured against the same nlatic group is involved at roonl tenlperature protein solution of pH 7.09 as reference. and that these two groups are likely not When a solution at pH 3.57, .5.62, or 8.00 involved at 9Oo.3When tyrosyl group ionizes was used in the proper compartment at room at alkaline pH, then the peaks at 29.5 and 245 nip appear. The spectruln below 27;i 111~ temperature (23 =t a’), there was essentially no observable spectrum in the region 240- in Fig. 2 indicates the possible involvenlent $50 mp. Two peaks at 29.5 and 245 nip be- of phenylalanyl and histidyl groups at high tenlperature. Since little is known about the came evident as pH of the protein solution perturbation of phenylalanyl and histidyl increased to 9.16. Figure 1 is a typical differgroups and since the possible decoluposition ence spectrum in alkaline pH where the magnitudes of 295 and 24.5 nip peaks in- of sonle urea at high tenlperature further no positive crease with increasing pH in the region of conlplicates the interpretation, tyrosyl ionization. The shape of I:ig. 1 and statenlent seenls justified at this tinle. It the ratio of the maxima at 245 and 295 111~ lnay be noted, however, that the difference are very close to the difference spectra of spectra of gluten, glutenin, and gliadin are tyrosiue and heme proteins reported by quitBe different from that of soybean trypsin Hermans (10) when a solution at high pH 3The tyrosyl groups ionixe normally (4). Alwas read against a solution of the same corn though the possibility remains that tyrosyl and ccntration at neutral pH. Figure 2 is a tryptophanyl groups might be involved in some typical difference spectrum at high temperainteractions not influenced by change of pH and ture. Roth solutions in Fig. 2 had the sanle by heating to 90’ such a possibility is not, very starting pH of 7.09. However the heated likely to exist. SEDIMENTATION COEFFICIENT or GLCTEN, GLUTENIN, AND GLIADIN IN 3 M VREA PLUS Bc-FPER AT ROOM TEMPERATCRE
438
WU AND
DIMLER
Wavelength, mp FIG. 1. Difference spectra of gluten at 25” in 3 ik! urea plus 0.11 IM KC1 plus 0.02 M buffer; pH 9.63 vs. pH 7.09 as reference. The optical density of the reference was set at zero at 350 mr. Gluten concentration was 2.37 mg. per milliliter in each solution. I
I
I
I
I
I
I
I
I
If
0.5 -
!
x Z .Y Z 0 0 I
k!b 250
I
300 Wavelength, mp
I
I
I
I
350
FIG. 2. Difference spectra of gluten at pH 7.97, 90.0”, in 3 M urea plus 0.11 M KC1 plus 0.02 M buffer vs. gluten at pH 7.09 (reference set at zero optical density at 350 mp), 30.5” in the same solvent. Gluten concentration 2.37 mg. per milliliter in both solutions.
inhibitor (12), where large tyrosyl and tryptophanyl peaks are evident at high temperature. Optical rotatory dispersion curves for gluten, glutenin, and gliadin were plotted by two methods. The first method was according to nloffitt (14):
value of X0 was taken to be 212 m/~ in accordance with that found best for poly--benzyl-L-glutamate in a variety of solvents (15). A typical Moffitt plot is shown in Fig. 3. The parameter bo was calculated from (Ml 100)[3/(n2 + 2)]/x04 times the slope of a plot of [a] (X2 - X,2) vs. l/(Xf - X,2). Since bo in Eq. (2) was small, the data were also plotted according to Drude equation (16): X2[a] = Xc$] + k.
where M is the average residue weight and n is the refractive index of the solvent. The
(3)
A Drude plot for gluten is shown in Fig. 4. The parameter X, was obtained from the slope of the straight line of X2[01]vs. [a]. A
CONFORMATIONAL
STUDIES
OF WHEAT
GLUTEN,
GLUTENIN
AND GLIADIN
-239
? = -30 x “do -32 I “2 -340 z?J
I 0 0-0, I 5
I
I
I 15
I 20
I @y-Q I 10
25
1
x 106 A= - A',
FIG. 3. A Moffitt plot of the rotatory dispersion of gluten in 3 111urea plus 0.09 M KC1 plus 0.04 llri borate at. room temperature, pH 10.1. Gluten concentrations 0.99 and 0.2252.
FIG. 4. A Drude plot of the rotatory dispersion of gluten in 3 M urea plus 0.09 M KC1 plus 0.04 iM borate at room temperature, pH 10.1. Gluten concentrations 0.99 and 0.22yc.
sumnary of the optical rotation data is given in Table III. Theoretically, the value for gluten should be a weighted average of glutenin and gliadin. However, the limited accuracv of the oolarimeter and the low solubility of gluteiin caused some apparent inconsistency of the gluten value from that of the average of glutenin and gliadin. The values in Table III, however, are accurate enough to make a qualitative statement and a relative comparison. The mall difference in values of bo , a0 , X, , and [CY]Wfor gluten and gliadin at high and low pH’s is within experimental error and considered not significant. There seems, however, a significant change in the optical rotatory parameters of glutenin between high and low pH. The same change in bo values was observed for glutenin in 8 M urea solution (17). If the gluten proteins can be approximated as a mixture of random coil and helix, then low
TABLE
III
OPTICAL ROTATORY PROPERTIES OF GLUTEN, GLUTENIN, AND GLIADIN IN 3 M UREA PLIX BUFFER AT ROOM TEMPERATCRE Wheat protein
Glut,en Gliadin
’ pH I 10.1 3.8 10.2
-40
4.4
Glutenin
10.3 4.2
-
-50
-622 -642
-108 -90 -52 -11
-688 -721 -596 -673
217 220 225 224 222 213
helical contents (perhaps below 20 Lil) are exhibited by all three proteins with gliadin having the highest value. The change in bo for glutenin at high pH is in the directiou of increasing helical content. Kretschmer (18) interpreted his optical rotatory dispersion measureulents according
440
WU AND
to Moffitt’s theory (15) and indicated that wheat gluten in 0.01 M formic acid and gliadin in 70 % ethanol have helical contents of about 35 %. Kretschmer’s results cannot be compared with ours because of the different solvents used. Glutenin has five disulfide groups per lo5 gm from amino acid analysis (5) ; disulfide-cleaved glutenin has a molecular weight of 20,000 and a much smaller intrinsic viscosity (19). An increase of intrinsic viscosity of glutenin was around pH 10 (Table I) ; this increase indicates that no disulfide bond is cleaved at least in the initial period when measurements were made. Glutenin showed a very large decrease in empirical electrostatic factor w at high pH from hydrogen in equilibria (5). Since w depends on molecular size, shape, and permeability at constant temperature and ionic strength, a decrease in w can result from an increase in molecular size, asymmetry, or permeability. For glutenin, at least, an increase in asymmetry of the molecule at high pH is supported by the following evidence, which includes: (1) An increase in intrinsic viscosity, (2) a decrease in sedimentation coefficient, and (3) a change in optical rotatory parameters in the direction of increasing helical content. Taylor and Cluskey (20) measured the intrinsic viscosities of glutenin and gliadin in sodium lactate buffer of pH 3.1 at ionic strengths 0.003 and 0.03. They concluded that glutenin behaves as a flexible, randomly coiled polyelectrolyte but that gliadin has a low, relatively constant, intrinsic viscosity, which perhaps indicates a comparatively globular protein. For gliadin, local electrostatic effect or an increase in permeability may cause a relatively small decrease in w at high pH from hydrogen-ion equilibria (5). Gliadin only shows small changes in viscosity, sedimentation coeficient, and optical rotatory parameters, and these changes border on experimental un-
DIMLER
certainties. Further physicochemical studies on glutenin and gliadin are planned to supplement the data available at present. ACKNOWLEDGMENT We thank sedimentation
G. E. Babcock for carrying velocity runs.
out the
REFERENCES 1. OSBORNE, T. B., “The Vegetable Proteins,” 2nd edition. Longmans, Green, New York, 1924. 2. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., Arch. Biochem. Biophys. 94,477 (1961). 3. WOYCHIK, J. H., BOUNDY, J. A., AND DIMLER, R. J., J. Agr. Food Chem. 9, 307 (1961). 4. Wu, Y. V., AND DIMLER, R. J., Arch. Biochem. Biophys. 102, 230 (1963). 5. Wu, Y. V., AND DIMLER, R. J., Arch. Biochem. Biophys. 103, 310 (1963). 6. JONES, R. W., TAYLOR, N. W., AND SENTI, F. R., Arch. Biochem. Biophys. 84,363 (1959). in 7. SCHACHMAN, H. K., “Ultracentrifugation Biochemistry.” Academic Press, New York, 1959. pH Determina8. BATES, R. G., “Electrometric tions.” Wiley, New York, 1954. Chem. 16, 341 9. YANG, J. T., Advan. Protein (1961). 1, 193 (1962). 10. HERMANS, JAN, JR., Biochemistry 11. TANFORD, C., SWANSON, S. A., AND SHORE, W. S., J. Am. Chem. Sot. 77, 6414 (1955). 12. Wu, Y. T’., AND SCHERAGA, H. A., Biochemistry 1, 995 (1962). A., “Protein Structure.” 13. SCHERAGA, H. Academic Press, New York, 1961. 14. MOFFITT, W., J. Chew. Phys. 25, 467 (1956). 15. MOFFITT, W., AND YANG, J. T., Proc. Nal.!. Acad. Sci. U.S. 49, 596 (1956). 16. YANG, J. T., AND DOTY, P. M., J. Anl. Chem. Sot. 79, 761 (1957). 17. ZOBEL, H. F., AND TAYLOR, N. W., In preparation. 18. KRETSCHMER, C. B., J. Phys. Chem. 61, 1627 (1957). 19. NIELSEN, H. C., BABCOCK, G. E., AND SENTI, F. R., Arch. Biochem. Biophys. 96,252 (1962). 20. TAYLOR, N. W., AND CLUSKEY, J. E., Arch. Biochem. Biophys. 97, 399 (1962).