Journal o f Protein Chemistry, Vol. 10, No. 1, 1991

Denaturation Behavior of Phaseolin in Urea, Guanidine Hydrochloride, and Sodium Dodecyl Sulfate Solutions S. S. Deshpande I and Srinivasan Damodaran 1'2

Received October 22, 1990

The denaturation behavior ofphaseolin in urea, guanidine hydrochloride, and sodium dodecyl sulfate solutions was examined by monitoring changes in the intrinsic fluorescence of tryptophan and tyrosyl residues. Changes in various fluorescence parameters, such as quantum yield, emission maximum, spectral half-width, fluorescence depolarization, and fluorescence quenching by acrylamide, have indicated that while phaseolin is relatively stable up to 8 M urea, it is completely destabilized in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate. Furthermore, while the denaturation ofphaseolin in urea solutions followed a two-step process, that in guanidine hydrochloride and sodium dodecyl sulfate followed a single-step process. While the accessibility of tryptophan residues to the nonionic acrylamide quencher is almost 100% in 6 M guanidine hydrochloride and 6 mM sodium dodecyl sulfate, only about 72% was accessible in 8 M urea compared to 52% in native phaseolin. The results presented here suggest that the protomeric structure of phaseolin is quite stable to changes in the environment. This structural stability may be partly responsible for its resistance to proteolysis by various proteinases. KEY W O R D S : Phaseolin; denaturation; structural stability; fluorescence.

protein of the c o m m o n dry bean. Phaseolin is a trimeric glycoprotein comprising three different subunits (a,/3, and y) of 51-53, 47-48, and 43-46 kD apparent molecular mass, respectively. It displays considerable sequence homology (over 70% when conservative substitutions are allowed) with pea vicilin and soybean #-conglycinin, the other two 7S homologous storage proteins found in food legumes. Among these three proteins, there are a total of 125 absolutely conserved residues with certain sections of the sequences more highly conserved than others. Yet, c o m p a r e d to vicilin and fl-conglycinin, phaseolin is extremely resistant to prateolysis both in vitro and

1. I N T R O D U C T I O N The underutilization of food legume proteins is often attributed to the resistance to proteolytic digestion of their major globulin fractions. As compared to animal proteins, those from various legumes are only 50-60% biologically available. Several studies have demonstrated that neither the deficiency of sulfur amino acids nor the presence of heat-labile protease inhibitors are the only reasons for the low biological value of legume proteins. These evidences p r o m p t e d Liener (1976) to suggest the unique structural features of legume proteins as major causes for their p o o r biological value. In recent years, most studies on the digestibility of legume proteins have been directed toward phaseolin, the major storage

in vivo.

Earlier studies have related such differences in their proteolytic behavior to certain key differences in their physicochemical and solution conformational states ( D e s h p a n d e and D a m o d a r a n , 1989a, 1990). We have earlier demonstrated that each subunit of phaseolin is composed of two highly stable domains

1 Department of Food Science, University of Wisconsin, Madison, Wisconsin 53706. 2 To w h o m all correspondence should be addressed.

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Deshpande and Damodaran

3. RESULTS The corrected emission spectra for total protein and tryptophan fluorescence of phaseolin are shown in Fig. 1. The /~max of the protein fluorescence spectrum of native phaseolin was blue shifted by about 3 nm as compared to its tryptophan fluorescence spectrum (Table I). The half-maximal spectral width (AA~/2) and the ratio parameter (r) were, respectively, 51 nm and 1.576 for the native protein. The fluorescence emission maxima of 329-332 nm for native phaseolin indicates that the fluorescent tryptophan residues are in a relatively hydrophobic environment. The relative fluorescence below 340 nm was greater with excitation at 260 nm than with 295 nm (Fig. 1). The latter wavelength induces fluorescence primarily from tryptophan, whereas 260 nm excitation elicits fluorescence from both tryptophan and tyrosine. In agreement with previous studies (Teale, 1960), the relative quantum yield observed for the two spectra was greater for the longer wavelength of excitation (Table I). Both protein and tryptophan fluorescence intensity increased by about 10-15% in up to 2 M urea concentrations (curves not shown in Fig. 1).

A

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Fig. 1. Corrected emission spectra of phaseolin in the presence of various concentration of urea. A - D refer to total protein emission spectra in 0, 4, 6, and 8 M urea, respectively, whereas a-d refer to tryptophan emission spectra at these concentrations. Protein concentration was 0.112 mg/ml.

However, it gradually decreased thereafter with further increases in urea concentrations. In 8 M urea, the protein fluorescence spectrum was characterized by a large red shift of about 27 nm as c o m p a r e d to

Table I. Fluorescence Characteristic of Native and Denatured Phaseolin Parameter a Xmax (nm) 260 nm Ex b 295 nm Ex b AAI/2 (nm) r

qbp qb Acrylamide quenching K~v (M 1) KQ (M -a) face Degree of polarization (P)

Tyr Trp d

Trp e

Native

8 M urea

6 M GuHC1

6 mM SDS

329.7 ± 1.2 332.2 ± 1.2 51 1.576 0.0960 (0.0009) C 0.1010 (0.0012) c

356.6 ± 0.7 346.1 ± 1.7 77 1.956 0.1301 0.0703

351.2 ± 2.4 353.9 ± 1.4 71 1.196 0.0561 0.0395

312.6 ± 1.7 338.9 + 1.5 62 0.915 0.684 0.0443

1.98 5.56 0.521

3.61 6.14 0.718

7.93 6.46 1.099

6.32 7.12 0.958

0.180 0.241 0.235

0.126 0.170 0.122

0.103 0.088 0.077

0.127 0.174 0.157

0.128 0.175 0.170

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Anisotropy

Tyr Trp d, Trp e

a See Materials and Methods section for the definitions of individual parameters. b Mean + standard deviation of 10 scans. c Value in the parenthesis is standard deviation of duplicate determinations at two different protein concentrations. d 295 nm excitation, emission monitored at 335 nm. e 295 nm excitation, emission monitored at 355 nm.

4.80

nm

Structural Stability of Phaseolin

107

that of the native protein, whereas the red shift was only about 1 4 n m for the tryptophan fluorescence spectrum. Under the experimental conditions, aqueous L-tryptophan at p H 7.0 had a m a x i m u m at 354.1 +0.9 nm. The protein emission spectra in 6 M and higher concentrations of urea also showed a distinct shoulder around 305 nm, indicating a significant contribution of tyrosyl fluorescence to the total emission spectrum. The effects of urea on various emission spectral characteristics of phaseolin are shown in Fig. 2. The fluorescence emission m a x i m a of both protein and tryptophan fluorescence spectra showed a gradual red shift with major changes occurring above 4 M urea. A similar pattern was also observed for AA~/~. The ratio parameter r gradually decreased up to 5 M urea concentration indicating an increase in tyrosyl fluorescence. However, above 5 M urea concentration, the ratio p a r a m e t e r increased reaching a m a x i m u m value of 1.956 in 8 M urea. This represented

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changes occurred in 4-5 M GuHCi v,,'L,,e:-~th :d shift was about 15 nm of the ~otal 20 nm sbik, '-[hc half-maximal spectral width increased from 5 i--71 nm in 5 M GuHC1 and remained unchanged thereafter. Due to the emergence and increasing contribution of the tyrosyl fluorescence to the protein fluorescence, the ratio parameter decreased by about one third. The changes in both @p and @Tw paralleled those in emission maxima, with major decreases in the quantum yields occurring in 3-5 M GuHC1. The @p and @W~pof phaseolin in 6 M G u H C I decreased by about 42 and 60%, respectively, as compared to the respective quantum yields of the native protein (Table I). The protein emission spectra of phaseolin (260 nm excitation) in SDS were remarkably different as compared to those in either urea or G u H C l (Fig. 5). The spectra were blue-shifted by about 18 nm as compared to the native protein spectrum, the emission, m a x i m u m being 312 nm in 6 m M SDS (Table I). On the contrary, the tryptophan emission spectra showed a red shift. However, the red shift of about 7 nm w~?~

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(K~v) constant using least-squares regression techniques from the initial linear portion of the curve, was 1.98 M -1. The effective quenching constant KQ for acrylamide quenching determined from the modified Stern-Volmer plot was 5.56 M -a, whereas the fraction of tryptophan residues accessible to acrylamide quenching was 52.1% for the native protein (Table I). The slopes of the Stern-Volmer curves gradually increased in increasing concentrations of both SDS and GuHC1 (Fig. 7), indicating easy accessibility of the fluorescent tryptophan residues to acrylamide as the denaturation proceeds. On the other hand, the downward curvature of the Stern-Volmer plot in 4 M urea suggested that a significant amount of fluorescence was retained due to the inaccessibility of at least some of the tryptophan residues to acrylamide. In 8 M urea, the Stern-Volmer plot actually showed an upward curvature indicating that static quenching was playing a major role in higher urea concentrations.

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Fig. 7. Stern-Volmer plots for acrylamide quenching oftryptophan fluorescence of phaseolin in SDS, GuHCI, and urea. For the sake of clarity, data at only selected concentrations of each denaturant are shown here. Fo and F are the tryptophan fluorescence intensities in the absence and presence of an indicated amount of quencher.

The behavior of the three fluorescence quenching parameters used to describe the acrylamide quenching of the intrinsic tryptophan fluorescence of phaseolin as a function of denaturant concentration is summarized in Fig. 8. The Stern-Volmer constant Ksv remained unchanged up to 3 M urea and then increased with further increase in urea concentration (Fig. 8A). On the contrary, the effective quenching constant KQ showed a reverse bell-shaped pattern. The fraction oftryptophan residues accessible to acrylamide increased up to a maximum of 82.3% in 5 M urea and then decreased with further increases in the urea concentration. The behavior of the Ksv and KQ quenching constants in GuHC1 (Fig. 8B) and in SDS (Fig. 8C) was similar to that observed in urea. However, unlike in urea, the fluorescent tryptophan residues in both these denaturants were fully accessible to acrylamide. Changes in Ksv were minimal up

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to 4 M GuHC1, and then it increased by almost fourfold as the concentration was increased to 6 M. The decrease in face in 1 M GuHC1 concentration was perhaps related to the increased ionic strength of the solution. At higher ionic strengths, many globular

proteins are known to become more compact, and this conformational change may be reflected in a lower accessibility of its fluorescent tryptophan residues to a given quencher. The accessibility of tryptophan residues to acrylamide showed a biphasic

Structural Stability of Phaseolin behavior. The initial increase in f, cc was observed between 1-2 M GuHC1 and probably was related to the dissociation of the three subunits of phaseolin protomer. The second and more pronounced increase observed above 3 M GuHC1 may reflect the unfolding of the tertiary structure of the individual subunits. The critical concentration of SDS in which maximal changes in the three quenching parameters were observed was in the range of 1.5-4.5 mM (Fig. 8C). The addition of denaturants caused a decrease in observed polarization values and hence, anisotropy of both tryptophyl and tyrosyl residues of the protein (Fig. 9A-C). However, significant differences were observed with respect to the individual denaturant. The extent of depolarization in both tyrosyl and tryptophyl side chains in urea and SDS was similar (Table I), the maximum decrease being approximately 31% in each case. On the contrary, in 6 M GuHCI, the respective decrease in tryosyl and tryptophyl anisotropy was about 45 and 65%. The greater drop in anisttropy values in GuHC1 as compared to SDS and urea suggests a more flexible structure for the side chains of these two amino acids, and thus a greater randomization of the tertiary and quaternary structures of phaseolin in 6 M GuHC1. The decrease in anisotropy was also a function of the denaturant concentration. For example, for tryptophan, depolarization essentially took place in 1.5-4.5 mM SDS concentrations, and the polarization values remained fairly constant in up to 12.5 mM SDS. In both urea and GuHC1, polarization values showed a gradual but insignificant decrease up to 4 M denaturant concentration followed by a steep decline in 4-6 M concentration range. The polarization value for tryptophan increased slightly in 8 M urea (Fig. 8A). The depolarization of tyrosyl side chains followed a similar pattern with respect to the denaturant concentration. 4. D I S C U S S I O N Phaseolin represents almost half of the total seed protein in dry beans. The nutritional value of bean proteins is thus largely determined by the extent that this protein is biologically available. Earlier studies have supported the hypothesis that the tertiary and quaternary conformations, as well as relative compactness of phaseolin and other homologous 7S storage proteins of soybeans and field peas, are important determinants of their proteolytic rates and thus of their relative biological availability (Deshpande and Damodaran, 1989a, b, 1990). Parameters

111 which are able to assess the conformational deformability of these proteins are therefore useful for defining the conditions, as well as the elimination of the conformational constraints necessary to improve their digestibility. A physical parameter, such as fluorescence emission, would therefore be of considerable importance in establishing in a biochemical perspective the nutritional properties and thus the biological availability of these proteins. A

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112 Phaseolin in neutral aqueous solution has a fluorescence spectrum typical of a class II protein tryptophan (notation of Burstein et al., 1973). The emission maximum of 332 nm suggests that its fluorescent tryptophan residues are buried in the interior of the protein. Earlier studies have also shown that they are not accessible to surface ionic quenchers (Deshpande and Damodaran, 1990). The tyrosine fluorescence was also evidenced in native phaseolin by the fact that a slight but definite hypochromic shift in emission maximum was observed with 260 nm excitation compared with 295 nm excitation. The presence of tyrosine fluorescence in native phaseolin suggests a lack of very efficient energy transfer from tyrosine to tryptophan. In the presence of the denaturants, tryptophan emission maxima shifted toward longer wavelengths. The extent of this red shift was of the order GuHC1 > u r e a > S D S . GuHC1 was thus more effective in denaturing the protein compared to the other two denaturants. The red shift in emission maxima was also accompanied by an increase in the half-maximal spectral width of the fluorescence spectra. This increase was largely due to the emergence and contribution oftyrosyl fluorescence to the protein spectrum. The increase in the spectral band width for SDS, however, was only half as much as in GuHCI or urea. Globular proteins, especially such as phaseolin which is a predominantly/3-sheeted protein, assume helical conformation in the SDS environment. Thus, phaseolin denaturation in SDS may be viewed as a "random-ordered" rather than a "random-coil" structure. Similarly, the tryptophyl side chains may be involved in mixed-micelle formation with the aliphatic chains of SDS thereby still maintaining a hydrophobic environment around them. This probably was the reason that the emission maximum of phaseolin in SDS was never larger than 339 nm as compared to 354nm for free tryptophan in water observed in the present study. The protein fluorescence spectrum of phaseolin in SDS was also characterized by a very large hypochromic shift as compared to the longer wavelength shifts observed in both urea and GuHC1. This blue shift was further accompanied by a ratio parameter r of less than unity, indicating that the tyrosyl fluorescence was a dominant factor. This also suggests a very low energy transfer from tyrosine to tryptophan in the presence of SDS. It is also quite likely that the tryptophan fluorescence may actually be quenched in the presence of SDS, thus resulting in an apparent blue shift of the emission maximum. The less efficient energy transfer from

Deshpande and Damodaran tyrosine to tryptophan and thus the decrease in r values in the presence of increasing denaturant concentration may also be related to an increase in the critical transfer distance for these two amino acids (estimated to be between 12.5-26/~) as the protein unfolds, thus separating the tyrosyl fluorescence from that of tryptophan. Among the three denaturants, only GuHC1 was effective in resolving the fluorescene spectrum into two distinct peaks corresponding to tyrosyt and tryptophan residues. The double emission peak in GuHC1 was evident at concentrations of 5 M and above. On the other hand, tyrosyl contribution to the protein fluorescence spectrum in both urea and SDS was evident only as a shoulder at 305-310 nm. In general, the changes in Stoke's shift and spectral band widths were also paralleled by decreases in the quantum yields and polarization of both tyrosyl and tryptophyl side chains of the protein. The critical concentrations of the three denaturants (where most of these spectral changes were observed) were 2-5 M for GuHCI, 4-6 M for urea, and 1.5-3 mM for SDS. At either end of these concentrations, most parameters remained unchanged, indicating that most conformational changes involving protein denaturation essentially took place within this critical range of denaturant concentrations. The tryptophan quantum yield of 0.101 for native phaseolin was similar to that reported for several proteins (Burstein et al., 1973; Kronman and Holmes, 1971). Both tryptophan and protein quantum yields decreased with increasing extent of protein denaturation. The decrease is usually related to the solvent quenching of the exposed fluorescent tryptophan residues. However, the extent of decrease in • was characteristic of each denaturant and was of the order G u H C I > S D S > u r e a , once again indicating that GuHC1 was a more effective denaturant of phaseolin. It is difficult to compare the changes in quantum yields of various native proteins and their denatured states, since there is no general consensus in the literature. The quantum yield of the denatured proteins is often influenced by the amino acid sequence around a tryptophan in a disrupted peptide chain and the "residual" three-dimensional structure that may persist in reduced denatured proteins. The higher values for both qbvrpand ~p for phaseolin in 8 M urea appears to be due to an incomplete or partial denaturation of its tertiary and quaternary structures. The Stern-Volmer plot for acrylamide quenching of intrinsic tryptophan fluorescence o f native phaseolin was characterized by a downward curvature indicative of the presence of multiple emissive

Structural Stability of Phaseolin components. The absence of upward curvature further suggested that the static quenching parameter, V, was very small. The accessibility of the fluorescent tryptophan residues to acrylamide in native phaseolin was, however, only 52%. Acrylamide is a nonionic polar compound that quenches the fluorescence of indole derivatives by a collisional process (Eftink and Ghiron, 1976). Since there are no obvious stereochemical requirements for this quenching process, the efficiency of the reaction is near unity. Thus, acrylamide has the ability to quench any excited tryptophyl residues that it happens to collide with regardless of whether the residue is located on the surface or the apolar interior of the protein. Acrylamide quenching therefore can yield information on a local or large-scale conformational fluctuations of the protein. That only half of the fluorescent tryptophan residues in native phaseolin was accessible to acrylamide suggests that the nanosecond time-scale fluctuations in the tertiary and quaternary structures of phaseolin are minimal due to its very high degree of compactness and stability imparted by strong hydrophobic and hydrogen-bonding interactions. Furthermore, it is also quite likely that at least some of the fluorescent tryptophan residues may be deeply buried in the interior of the phaseolin protomer and sterically shielded by surrounding protein segments. In such a case, the frequency of their collision with acrylamide will be reduced and thus its accessibility. For phaseolin, the extent of steady-state collisional quenching measured as Fo/F was fairly similar in SDS and GuHC1, whereas the fluorescence decrease in urea solutions was consistently lower by 50-60% at all the concentrations studied. In fact, at 0-0.2 M acrylamide concentrations, no more than 30-35% fluorescence was quenched in 1-6 M urea solutions, whereas a maximum quenching of 40-45% occurred only at 6-8 M urea concentrations. On the contrary, over 55% fluorescence was quenched at 0.15 M acrylamide in both SDS and GuHC1 concentrations where significant changes in most other spectral properties were also observed. This behavior was also reflected in the experimentally determined K~v values from the initial linear portions of the Stern-Volmer plots. The K~v value in 8 M urea was almost half that observed in GuHC1 and SDS. Furthermore, the fraction of fluorescent tryptophan residues accessible to acrylamide was only 70% of that accessible in either GuHC1 or SDS. The linearity of Stern-Volmer plots in SDS and GuHC1 also suggested that the fluorescence of tryptophan residues was readily accessible to acrylamide.

113 The downward curvature of the Stern-Volmer plot was still evident in 4 M urea indicating that some of the fluorescent tryptophan residues might be completely inaccessible to acrylamide. Alternatively, a large difference in lifetime for the various fluorophores, rather than the rate constant, might also explain the selectivity in quenching. However, unlike in SDS and GuHC1, the Stern-Volmer plot in 8 M urea above 0.2 M acrylamide showed an upward curvature diagnostic of static quenching. Such positive deviation from linearity upon unfolding of pepsin and 13-trypsin to acrylarnide has been known, whereas among the multiple tryptophan-containing proteins studied so far, only native bovine serum albumin shows the static quenching parameter (Eftink and Ghiron, 1976). Although the assignment of the collisional and static components is straight forward with single tryptophan proteins, for multiple tryptophancontaining proteins, it is not a priori obvious to what extent quenching occurs by these two kinetic processes. In the latter case, the static component is often masked and the K~v parameter may be considered to be a collisional quenching constant equal to Kq(em %, where Kq(e~ ) is a crude estimation of the average exposure of the fluorescing residues in the protein and % is the average lifetime (Lehrer, 1971). Therefore, the dynamic quenching constant K~v can be used directly only if the fluorescence lifetimes of the molecules to be compared with each other (in our case the extent of phaseolin denaturation in the three denaturants studied) are similar. Eftink and Ghiron (1981) further interpret the contribution of static quenching to the overall process as indicative of a single population of fluorophores which interact with the quencher with nearly identical quenching constants. If such is the case, then the analysis of the quenching data by the modified Stern-Volmer equation of Lehrer (1971) would suggest a single population of reactive groups, since a linear plot extrapolating to unity will be obtained. The dynamic and the effective collisional quenching constants (Ksv and Ko, respectively) will therefore be in close agreement. However, for phaseolin in 8 M urea, these values differed by almost two-fold, indicating a substantial contribution of the static quenching in 8 M urea. In general, the increase in the dynamic quenching constant Ksv with denaturant concentration in the present study indicates the increased collisional frequency between the fluorescent tryptophan residues of phaseolin and the quencher molecules as the protein is being unfolded. This change in Ksv was

114 paralleled by a corresponding increase in the accessibility of the tryptophan residues to the solvent. However, in the presence of urea, a biphasic behavior could be observed for both the dynamic quenching constant and the face. In the first case, the first transition could be observed between 3 and 6 M and a second one at 6-8 M urea, whereas for the latter, these transitions occurred between 2 and 5 M and 5 and 8 M urea concentrations. We can only speculate as to the reason for the decrease in tryptophan accessibility at high urea concentrations. It may be due to either a partial unfolding of the phaseolin subunits or that the partially or completely unfolded subunits may later reassociate to form aggregates. Such aggregation would effectively minimize the collisional frequency between the quencher molecules and at least a fraction of the fluorescent tryptophan residues in 8 M urea. Indirectly, the evidence also comes from the fact that the /~maxof the tryptophan fluorescence spectrum of phaseolin in 8 M urea was only 346.1 nm as compared to 353.9 nm in 6 M GuHCI and 354.1 nm for free L-tryptophan under our experimental conditions. This also indicates that not all the tryptophan residues ofphaseolin are being exposed to the solvent in 8 M urea. Furthermore, the polarization values for tryptophan in 8 M urea were also almost twice as much as were observed in 6 M GuHCI indicating less randomization of its side chains in 8 M urea. In addition, the quantum yield of tryptophan qb-rrp in 8 M urea was 44% higher compared to that in 6 M GuHC1. Since the quantum yields are generally normalized upon denaturation of the protein, it also supports the contention that either an incomplete unfolding or the aggregation of the unfolded phaseolin subunits might have occurred. The extent of phaseolin denaturation in various denaturants was also monitored by following changes in the polarization of both tyrosyl and tryptophan environments. Polarization is a sensitive index of overall protein shape. Phaseolin has a relatively high polarization value for its tryptophan side chains (0.241). This is consistent with the compactness of the protein and a relatively lower accessibility of its tryptophan fluorescence to acrylamide. The tryptophyl side chains may be expected to have less freedom of rotation in the interior of the protein matrix compared to when they are exposed to solvent. As compared to tryptophan, the tryrosyl side chains in native phaseolin seemed to have a greater degree of rotational freedom. Approximately two thirds of the total tyrosyl residues in native phaseolin are either partially or completely accessible to solvent (Desh-

Deshpande and Damodaran pande and Damodaran, 1990). This is consistent with a low polarization value of 0.180 for tyrosine. Under increasingly denaturing conditions, the degree of polarization and thus the anisotropy of both aromatic amino acids falls precipituously by 40-70% of the native values, suggesting (though in the absence of lifetime data, not proving) that the protein conformation is being effectively randomized. The polarization data also suggested GuHC1 to be a more effective denaturant of phaseolin than either urea or SDS. The extent of depolarization was similar in both urea and SDS. However, these two may reflect totally different local environments around the side chains of the aromatic amino acids. While the lower degree of depolarization in urea may be related to the incomplete unfolding of the phaseolin protomer, that in SDS may reflect the formation of mixed micelles around the side chains of tryptophyl residues. Such interactions in the latter case may also lower the available rotational freedom of the tryptophan residues. It should, however, be noted that the decrease in polarization or anisotropy does not necessarily reflect a simple average of the rotational motions, unless all tryptophan residues undergo homogeneous motions or have the same fluorescence lifetimes (Kouyama et al., 1989). Since phaseolin is composed of three subunits in its native conformation, denaturation implies dissociation of the three subunits, as well as randomization of the conformation of each constituent subunit. The question arises if it is possible to observe these two events independently. Acrylamide quenching data in GnHCI suggest that while the dynamic quenching constant K~v had changed little up to 4 M GuHC1, the accessibility of the tryptophan residues had increased by almost 50% in 2 M GuHC1. We have earlier shown that at least some of fluorescent tryptophan residues in phaseolin are involved in subunit interactions (Deshpande and Damodaran, 1989c). Thus, our present data indicate that the dissociation of phaseolin subunits apparently begins in 2 M GuHCI thus exposing the tryptophan residues involved in subunit interactions to aqueous solvent. However, changes in other emission spectral characteristics, such as Amax and quantum yields, are minimal up to 4 M GuHC1 concentration. The environment a r o u n d the fluorescent tryptophan residues above 4 M GuHC1 changes concomitantly with the appearance of the tyrosine emission, a significant reduction in the quantum yield, as well as increased depolarization of both tyrosyl and tryptophan side chains. This second set of events is thus

Structural Stability of Phaseolin probably related to the unfolding of the individual subunit chains of phaseolin. On the other hand, dissociation, as well as unfolding of phaseolin subunits in SDS, appears to take place simultaneously, since changes in all the spectral properties took place within a narrow concentration range of 1.5-3 nM SDS. Changes in emission spectral properties of phaseolin in urea solutions were more difficult to interpret, since even in 8 M urea unfolding of phaseolin subunits appeared to be still incomplete. This contention was supported by the fact that the quantum yield of fluorescence, as well as depolarization of tryptophan side chains in 8 M urea, still has not reached the limiting values as were observed in 6 M GuHC1. Furthermore, since denaturation increases the distance between the average tyrosyl and trytophan residues, thereby rendering energy transfer less efficient, the ratio parameter r would be expected to decrease. However, the conformation of phaseolin in 8 M urea appeared to have a greater energy transfer between these two residues as compared to that in native phaseolin. Thus, the protomeric structure of phaseolin in urea appears to be quite stable.

115 REFERENCES Brand, J. G., and Cagan, R. H. (1977). Biochim. Biophys. Acta 493, 178-187.

Burstein, E. A., Vedenkina, N. S., and Ivkova, M. N. (1973). Photochem. Photobiol. 18, 263-279.

Deshpande, S. S., and Damodaran, S. (1989a). J. Food Sci. 54, 108-113.

Deshpande, S. S., and Damodaran, S. (1989b). Biochim. Biophys. Acta 998, 179-188.

Deshpande, S. S., and Damodaran, S. (1990). Int. J. Peptide Protein Res. 35, 25-34. Eftink, M. R., and Ghiron, C. A. (1976). Biochemistry 15, 672-680. Eftink, M. R., and Ghiron, C. A. (1981). Anal. Biochem. 114, 199-227. Hall, T. C., McLeester, R. C., and Bliss, F. A. (1977). Plant Physiol. 59, 1122-1124. Kronman, M. J., and Holmes, L. G. (1971). Photochem. Photobiol. 14, 113-134. Kouyama, T., Kinosita, K., Jr., and Ikegami, A. (1989). Eur. Z Biochem. 182, 517-522. Lehrer, S. S. (1971). Biochemistry 10, 3254-3267. Liener, I. E. (1976). J. Food. Sci. 41, 1076-1081. McGuire, R., and Feldman, I. (1975). Biopolymers 14, 335-351. Rajkowski, K. M., and Cittanova, N. (1981). J. Theoret. Biol. 93, 691-696. Teale, F. W. J. (1960). Biochem. J. 76, 381-388. Teale, F. W. J., and Weber, G. (1957). Biochem. J. 65, 476-482.