a proposed mechanism for the cryoaggregation of the seed storage globulin and its polymerized form...

A PROPOSED MECHANISM FOR THE CRYOAGGREGATION OF THE SEED STORAGE GLOBULIN

AND ITS POLYMERIZED FORM FROM TRITICUM AESTIVUM

MASSIMO F. MARCONE' and RICKEY Y. YADA

Department of Food Science Ontario Agricultural College

University of Guelph Guelph, Ontario CANADA NIG 2WI

Received for, Publication on June 2 I , 1994 Accepted for Publication on November 7, 1994

ABSTRACT

The pur@ed salt-soluble globulin and its polymerized (i. e. , polymerized via interchain disulfide bonds between various subunits of differing molecular weights, excluding any interchain disulfide bonds between the 35,000 and 49,000 Da subunits) counterpan from wheat (Triticum aestivum) seed were studied at a variety of temperatures and holding times (i. e. , 25. 15, 1OC for holding times between 0 and 600 min). A mechanism responsible for cryoaggregation is proposed. The polymerized form of the globulin was found to be much more cryoaggregatable than the nonpolymerized form. Ultraviolet (UV) and circular dichroism (near- and far- UV) spectral analyses revealed that the polymerized globulin was more susceptible to conformational changes than its nonpolymerized form with decrease in temperature. It is suggested that the higher flexibility of the polymerized globulin would allow for subtle changes in protein conformation, and therefore, permit the surface aromatic amino acids to interact with other hydrophobic groups on neighboring protein molecules in a cooperative manner. The latter situation may result in aggregation and precipitation.

INTRODUCTION

Since the classical work of Osborne (1907), which classified wheat proteins according to their solubility characteristics, much physicochemical information

'To whom correspondence should be sent. Journal of Food Biochemistry 18 (1995) 147-163. All Rights Reserved. 0 Copyright 1995 by Food & Nutrition Press, Inc., Trurnbull, Connecticut.

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147

148 M.F. MARCONE and R.Y. YADA

has been acquired about the two most important wheat storage proteins, i.e., the prolamins and glutelins. Comparatively few studies have focussed on the two soluble proteins, i.e., the albumins and the globulins, although it has been shown that they are partly responsible for differences in baking characteristics observed among flours (Pence et al. 1954).

Okada et al. (1987) found that the proportion of polymerized globulin increased during mixing and its nonpolymerized and polymerized forms might play an im- mrtant role as binders during dough mixing. They further found that the globulin together with the other flour proteins were susceptible to cryoprecipitation and it was postulated that this phenomenon was due to the exposure of hydrophobic groups.

Cryoglobulins were first discovered in 1933 in human blood serum and other biological fluids and were defined as those immunoglobulins cryoprecipitable at temperatures around 4-5C. Cryoprecipitation was found to be a reversible event (Kosarev et al. 1984).

In the medical field, temperature sensitive proteins have been associated with such diseases as rheumatoid arthritis and other autoimmune diseases. Classical symptoms of cryoglobulinemia include Raynaud’s syndrome with gangrene of chilled body parts and hyperviscosity syndrome causing renal lesions and lesions of the central nervous system (Kosarev et al. 1984). Although the clinical significance of cryoglobulins is evident, few studies except for that of Kosarev ef al. (1984) have been performed in order todetermine the mechanism by which cryoprecipitation occurs. As early as 1962, attention was focussed on the similarity that exists between the immunological cryoglobulins and that of the so-called “plant cryoproteins” (Ghetie and Buzila 1962).

It was, therefore, the purpose of this paper to study at the molecular level the physico-chemical properties of the wheat globulin(s) and to propose a mechanism which may in part be responsible for their reported cryoaggregation.

MATERIALS AND METHODS

Sample Preparation

As described in Marcone and Yada (1995).

Protein Fractionation and Protein Concentration Determination

As described in Marcone and Yada (1995).

MECHANISM FOR THE CRYOAGGREGATION 149

Protein Isolation and Purification

Isolation and purification of the two globulin fractions were performed as described in the gel filtration and anion-exchange chromatography sections in Mar- cone and Yada (1995).

UV Spectroscopy

UV absorbance scans were generated using a Shimadzu UV-260 recording spectrophotometer (Tekscience, Mississauga, ON) between the wavelengths of 240 and 310 nm. Protein (0.375 mg/ml) was dissolved in a 35.1 mM potassium phosphate buffer, pH 7.50, containing 0.4 M NaCl, filtered through a 0.22 p filter and allowed to equilibrate for 30 min at 22C prior to spectroscopy. Scans were performed in triplicate.

Near- and Far-W Circular Dichroism Spectroscopy (CD) Circular dichroic measurements were carried out in the near-UV (240-320 nm)

at 20C under constant nitrogen purge using a Jasco JaoO spectropolarimeter (Japan Spectroscopic Co. Ltd., Tokyo, Japan) with cell path length of 10 mm. A protein concentration of approximately 0.1 m g / d in 35.1 mM potassium phosphate buffer, pH 7.5, containing 0.4 M NaCl was used in the determinations. Circular dichroic measurements were carried out in the far-UV (190-250 nm) using a Jasco 5-600 spectropolarimeter (Japan Spectroscopic Co. Ltd., Tokyo, Japan) with a cell path length of 1 mm. Secondary structure fractions were determined using the Jasco SSE program, which is based on the algorithm of Chang et al. (1978), and the data base of Hennessey and Johnson (1981). Analyses were performed in triplicate with six scans per replicate.

Surface Hydrophobicity (So)

Aromatic surface hydrophobicity of the globulins were determined using the hydrophobic probe, 1 -anilino-8-naphthalene sulfonate (ANS), according to the methods of Hayakawa and Nakai (1985) and Akita and Nakai (1990), using protein concentrations between 0.025 and 0.19 mg/ml. An excitation wavelength of 380 nm and emission wavelength of 475 nm were used. Samples were equilibrated for 20 min at their respective temperatures, i.e., 25, 15, and lOC, and measurements taken promptly at the assigned temperatures. Analyses were performed in triplicate.


Quantitative Determination of Sulfhydryl and Disulfide Groups

Sulfhydryl and disulfide groups were quantitatively determined as described by Beveridge et al. (1974), using 5,5 ‘-dithiobis-2-nitrobenzoic acid (Ellman’s reagent). Analyses were performed in triplicate.

Spectroscopic Cryoaggregation Profiles

Protein solutions of 0.375 mg/ml in the buffer described earlier were equilibrated at temperatures between 25 to 3C for various lengths of time (i.e., 0, 15, 30, 60, 120,600 min) and their turbidity measured at 600 nm. A Shimadzu UV-260 dual-beam recording spectrophotometer (Tekscience, Mississauga, ON) equip- ped with variable temperature control was used for the measurements. Analyses were performed in duplicate.

RESULTS AND DISCUSSION

According to Okada et al. (1987), the wheat globulin, as well as the other wheat proteins, are cryoaggregatable under the appropriate temperature conditions. Although it had been postulated that such a temperatursinduced aggregation was caused primarily due to an exposure of hydrophobic groups, no information with regards to the mechanism for such a phenomenon has been offered.

Cryoaggregation profiles (Fig. 1) showed that the polymerized globulin fraction (i.e., polymerized via interchain disulfide bonds between various subunits of differing molecular weights, excluding any interchain disulfide bonds between the 35,000 and 49,000 Da subunits) (fraction 2) was much more cryoaggregatable than fraction 3. It was noted that holding times up to 30 min at temperatures as low as 7C produced little precipitation; however, aggregation increased with longer holding times and lower temperatures (Fig. 1).

In order to determine if covalent bond formation (disulfide linkages) were con- tributing to the cryoaggregation of the globulin, N-ethylmaleimide (NEM) was incorporated into the buffer containing the protein. This blocking agent inhibits sulfhydryl-disulfide bond exchange by alkylating any free surface sulfhydryls, thereby, preventing their involvement in suithydryl-disulfide interchange. Cryoag- gregation of the globulin was not inhibited in the presence of NEM at concentrations of 0.3, 0.6, 1.0, 4.0 and 10.0 mM, the results indicate that sulfhydryl/ disulfide interchange was not involved in the aggregation phenomenon. The latter was also confirmed when excess /3-mercaptoethanol or dithiothreitol (both disulfide reducing agents) were incorporated into the buffers. Filtration, collection and analysis of the cryoaggregated globulin did not show any appreciable increase in the previously determined amounts of SH and S-S in the globulins (Marcone

MECHANISM FOR THE CRYOAGGREGATION 15 1

FRACTION 3

FRACTION 2

FIG. 1 . CRYOAGGREGATION PROFILES OF FRACTION 2 AND FRACTION 3 HELD AT A VARIETY OF TIMES AND TEMPERATURES

Analyses were performed in triplicate.


and Yada 1995). In fact, cryoaggregation was also observed to be reversible for each globulin, When they were gradually warmed to rmm temperature and held over an extended period of time (approximately three (3) h (data not shown)), the precipitate resolubilized. This reversibility indicates that noncovalent bonds were likely involved. The sue of the polymerized globulin had little effect on the observed differences in cryoaggregatability since the concentrations (i.e., 0.375 mg/ml) of the two globulins were kept the same. Fewer individual molecules of the polymerized protein in solution as compared to the smaller globulin leads to less possible collisions due to their slower movement due to brownian motion (Sisler et al. 1961). In fact, the greater number of the non-polymerized globulins and higher surface area to volume ratio would otherwise make it a more suitable candidate for aggregation.

A variety of analytical tests were conducted in an attempt to determine the im- portance, type and magnitude of the structural changes (if any) taking place within the two globulins with decrease in temperature and how they either induce or enhance the cryoaggregation of these proteins. To facilitate the systematic monitor- ing of these changes in protein conformation, various temperatures and holding times were selected, taking into consideration the temperatures and times for the onset of precipitation. Because UV spectroscopic methods and the interpretation of the results are adversely affected by the turbidity of the sample during analysis, studies below 1OC were not possible. To this end, three reference temperatures were selected, i.e. 25, 15, IOC, and a holding time of 20 min. It was our intent, therefore, that by studying some physicochemical properties at these temperatures that trends would be established which would allow us to propose a mechanism for cryoaggregation of the proteins.

Preliminary tests employing UV spectral absorption techniques showed that the absorption maxima and overall spectral pattern of globulin fraction 3 were very stable and consistent at all three selected temperatures, whereas globulin fraction 2 demonstrated a small but consistent blue shift (to shorter wavelengths) with decrease in temperature (data not shown). The differences in spectra observed for fraction 2 indicate a conformational change due to unfolding (Schmid 1990). Therefore, the above data indicate that globulin fraction 2, unlike fraction 3, was susceptible to subtle changes in tertiary structure with decreasing temperature.

Because near-UV circular dichroism (CD) spectra reflect changes in the tertiary structure of proteins (Strickland 1974) brought about by the interaction and orientation of the aromatic rings of tyrosine, tryptophan and phenylalanine with other amino acid moieties in the protein, near-UV CD spectra of the globulin fractions were investigated at the various temperatures. Near-UV CD spectral patterns for fractions 2 and 3 at 25C were quite different (Fig. 2), despite similarities in aromatic amino acid contents (see Table 1, Marcone and Yada 1994); such differences may indicate differences in motility of the surface aromatic amino


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TABLE I . CIRCULAR DICHROIC SECONDARY STRUCTURE OF THE VARIOUS FRACTIONS OF THE

PURIFIED GLOBULIN FROM TRITICUM A E W U M

Fraction 2' Fraction T 25C 15C 1OC 25C 15C 1OC

Fraction-

a-helix 26.4(+0.3) 23.4(k0.4) 26.3(+0.3) 8.0(+0.0) 8.0( 20.1) 9.4( kO.1)

B-sh-t 19.0( e0.3) 30.4( k0.4) 1 9 4 20.3) 53.4( 20.4) 54.1( 20.2) 48.9( k0.2)

8 - m 30.6(k0.1) 24.5(k0.4) 25.9( k0.7) 13.8( k0.2) 12.2(k0.2) 15.0( k0.3)

Random coil 24.8( 20.2) 25.7( k0.5) 26.q20.4)

'Expressed as a percentage of total bResults are the mean values (f standard deviation) of three replications 'Fractions 2 and 3 from the gel filtration column after further plrification on the anion exchange column

24.0( 20.1) 21.7( k0.4) 28.3( 20.6)

acids in the two fractions (Strickland 1974) which would be reflected in differences in flexibility of the protein backbone. Identification of the fine structure of the individual aromatic groups was difficult in view of the low intensities of the CD spectral scans. Tentative identification of the fine structure of phenylalanine for globulin fraction 3 at 25C was made at 258.0 and 264.0 nm, whereas the M)-cm- ' 'Lb of tryptophan was assigned to 290.2 nm and its 0 + 850-cm- ' 'Lb band at 286.4 nm. Tyrosine did not show any characteristic 0 + 800-cm- ' band around 275.0 nm (Fig. 2). For fraction 2 the 0 + 850-cm-' 'Lb band of tryptophan was assigned to 286.2 nm but lacked the O-O-cm- ' 'Lb band (Fig. 2). In contrast, tyrosine showed its characteristic 0 + 800-cm- ' band at 275.0 which was lacking in fraction 2 (Fig. 2). The fine structure for phenylalanine could not be identified.

When the temperature was decreased to 15 and lOC, globulin fractions 2 and 3 both showed general increases in the intensity or amplitude of their spectra (Fig. 3 and 4). This increase in amplitude for both globulin fractions is indicative of a change in tertiary structure due to aromatic amino acids becoming less motile or being in closer proximity to one another. In fact, at lower temperatures the molecular mobility of all amino acids, but especially the bulky aromatic amino acids, would be greatly reduced and enhance their ability to associate with one another through a variety of short, medium and long range attractive forces (Strickland 1974).

Secondary structure determination from CD spectral scans showed that fraction 3 had relatively high levels of the @-sheet fraction and comparatively low levels of a-helix (Fig. 5 , 6 and 7) (Table 1). This is in agreement with the results obtained by other researchers for the curcurbit globulin, edestin globulin, peanut globulin (Jacks er al. 1973), amaranth globulin (Marcone and Yada 1991) and


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the 11s soybean globulin (Jacks et al. 1973; Marcone et al. 1994). In contrast, the polymerized globulin (fraction 2) possessed relatively higher levels of a-helix, lower levels of 0-sheet and higher levels of &turn. Although both fractions varied in their organized structures, they had similar levels of random coil (Table 1).

Globulin fraction 3 showed very little change in CD spectra and secondary structure fractions with decrease in temperature. On the other hand fraction 2 showed considerable changes to its CD spectra below 200 nm (Fig. 7) and in its secondary structure with decreasing temperature. Most notable was the increase in 0-sheet at 15C and the drop in 0-turn and increase in random coil at 1OC in globulin fraction 2. Since @-turns are known to be composed of alternating hydrophilic and hydrophobic amino acid residues (Kanehisa and Tsong 1980) and usually are located on the surface of globular proteins (Kuntz 1972; Rose et al. 1985), it was postulated that the major conformational changes were occurring at the surface of the polymerized globulin. The above far-UV CD spectral data tend to indicate that fraction 2 was more prone to conformational change compared to fraction 3 with decreasing temperature, and tend to support conclusions drawn from UV and near-UV CD spectral data (i.e., fraction 2 being more flexible than fraction 3). Studies on the biological function of proteins have shown that interactions between proteins (i.e., protein - protein interaction) generally occur between their most flexible peptide regions (Alber et al. 1983).

A study of the surface aromatic hydrophobicity of each of the two globulins showed that each possessed the same level of hydrophobicity at 25C (Fig. 8). With decrease in temperature the surface hydrophobicity increased progressively for both globulins but the magnitude of change for fraction 3 was greater. The increased hydrophobicity seen for both fractions with decreasing temperatures may indicate that hydrophobic interactions were partially responsible for the structural stability of the two fractions such that progressive protein unfolding occurred at lower temperatures, thereby, exposing previously buried aromatic amino acids (Taborsky 1978). The increase in hydrophobicity measured with ANS, seen in fraction 2 as a function of decreasing temperature, however, may have been partially modulated (i.e., the extent of increase decreased) due to the higher flexibility of fraction 2 as compared to fraction 3. The binding of the ANS probe to the surface aromatic amino acids results in a complex with a higher degree of hydrophobicity which tends to bury itself, thereby decreasing the fluorescence emission more in a flexible protein (fraction 2) as compared to a more rigid protein (fraction 3). Ponnuswamy et al. (1982) stated that a high degree of cooperativ- ity exists in the stabilization of nonpolar residues. Conversely, for fraction 3, once the ANS probe bound to the surface aromatic amino acids it would be difficult to bury this complex due to the higher rigidity of this protein fraction; this results in a higher fluorescence emission as compared to fraction 2.


4

H Y d r 3 0 P h 0 b i 2 C i t Y

S ’ 0

0 25 15

Temperature ( C) 10

~~ 1 Fraction 3 Fraction a FIG. 8. ANS SURFACE HYDROPHOBICITY OF THE GLOBULIN

FRACTIONS AT 25, 15, IOC Analyses were performed in triplicate.

In conclusion, it was postulated that the potentially higher surface flexibility of fraction 2 compared to fraction 3 may help to explain the higher cryoaggregation for this fraction. The higher flexibility allows for subtle changes in protein conformation which allow for surface aromatic amino acids to interact in a more cooperative fashion with other hydrophobic groups on a neighboring molecule, and therefore initiate and enhance the aggregation process.


ACKNOWLEDGMENTS

The financial assistance of the Natural Sciences and Engineering Research Coun- cil of Canada is gratefully acknowledged.

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a proposed mechanism for the cryoaggregation of the seed storage globulin and its polymerized form...

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