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Fluorescence quenching and time-resolved fluorescence studies on Trichosanthes dioica seed lectin
Journal of Photochemistry and Photobiology B: Biology 80 (2005) 93–100 www.elsevier.com/locate/jphotobiol
Fluorescence quenching and time-resolved fluorescence studies on Trichosanthes dioica seed lectin Nabil Ali Mohammed Sultan, Musti J. Swamy
*
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India Received 21 January 2005; received in revised form 10 March 2005; accepted 10 March 2005 Available online 29 April 2005
Abstract Fluorescence quenching and time-resolved fluorescence studies have been carried out on the Trichosanthes dioica seed lectin (TDSL). The emission kmax of native TDSL, seen at 328 nm, shifts to 343 nm upon denaturation with 6 M guanidinium chloride. Quenching titrations were performed with neutral (acrylamide and succinimide) and ionic (I and Cs+) quenchers in order to probe the exposure and accessibility of tryptophan residues of the protein. Maximum quenching was observed with acrylamide, followed by succinimide, iodide and Cs+. Dramatic increase in the extent of quenching and other quenching parameters by all the quenchers were observed upon denaturation of TDSL, suggesting that all the tryptophan residues in native TDSL are buried in the hydrophobic core of the protein. Increase in the extent of quenching upon denaturation of TDSL was maximum with I and minimum with Cs+, suggesting the presence of positively charged residue(s), near at least one tryptophan residue. Addition of saccharide ligands such as methyl-b-D-galactopyranoside and lactose led to a small, but reproducible decrease in the fluorescence intensity of the lectin. The presence of lactose provided a partial protection against quenching by I, Cs+ and succinimide, but not acrylamide. In timeresolved fluorescence measurements the fluorescence decay curves could be best fitted to biexponential patterns with lifetimes of 4.09 and 1.53 ns for native lectin, 3.40 and 1.65 ns for the lectin in presence of 0.1 M lactose and 3.50 and 1.40 ns for denatured lectin. Ó 2005 Elsevier B.V. All rights reserved. Keywords: Intrinsic fluorescence; Agglutinin; Tryptophan; Acrylamide; Succinimide; Iodide ion; Cesium ion
1. Introduction Plant lectins are proteins with at least one non-catalytic domain which binds reversibly and specifically a mono- or oligosaccharide [1]. Lectins have elicited considerable interest due to a variety of interesting biological properties exhibited by them such as blood-group specific hemagglutination, mitogenicity, and the ability to distinguish between normal and malignant cells. In some plants, lectins may function as defense proteins whereas in others they may be responsible for the symbiosis between plant roots and nodulating Rhizobia. In * Corresponding author. Tel.: +91 40 2301 1071; fax: +91 40 2301 2460. E-mail address: [email protected] (M.J. Swamy). URL: http://202.41.85.161/~mjs/ (M.J. Swamy).
1011-1344/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.03.003
addition, lectins are also widely used in the detection, isolation and characterization of glycoconjugates and in the fractionation of cells for their use in bone marrow transplantation [2,3]. All these interesting properties, functions and applications are manifested through the specific recognition of carbohydrates by lectins. Trichosanthes dioica seed lectin (TDSL) is a galactose/ N-acetylgalactosamine-specific, heterodimeric glycoprotein with at least one intersubunit disulfide bridge and an apparent molecular mass of 55 kDa [4]. It exhibits a higher preference for b-galactosides over the corresponding a-anomeric derivatives. Chemical modification studies have implicated tyrosine residues in its carbohydrate binding activity. Circular dichroism spectroscopic studies indicate that its secondary structure is made of 10% a-helix, 37% b-sheet, 21% turns and the rest
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unordered chain [4]. TDSL is similar to several other seed lectins from Cucurbitaceae such as Trichosanthes kirilowii lectin, snake gourd (Trichosanthes anguina) seed lectin, and Trichosanthes cucumerina seed lectin, purified in this laboratory and elsewhere [5–7]. The intrinsic fluorescence of proteins arises primarily from the side chains of tyrosine and tryptophan residues. By exciting the protein sample at 295 nm or above, where tyrosine residues do not absorb, it is possible to study the fluorescence due to tryptophan alone [8]. Changes in the fluorescence characteristics of tryptophan residues are used widely to obtain information about conformational transitions in proteins, association of subunits in oligomeric proteins, protein unfolding or ligand binding to proteins [8–11]. The indole side chains of the tryptophan residues of TDSL could not be modified by N-bromosuccinimide under native condition and therefore appear to be buried in the protein matrix [4]. In order to investigate in greater detail the degree of exposure and environment of the tryptophan residues in TDSL, we carried out fluorescence quenching and time-resolved fluorescence studies on this lectin under native and denaturing conditions. The results obtained are presented here. 2. Materials and method
son, NJ, USA, website: http://www.jobinyvon.com). Slit widths of 3 and 6 nm were used on the excitation and emission monochromators, respectively. The integration time was set at 0.3 s. The calibration of the spectrometer was checked frequently using the Raman scattering band for water. The mcorrect feature in the operation software was routinely used during the spectral scan to correct for the photomultiplier characteristics. Absorption spectral measurements were made using a Shimadzu UV3101PC double-beam spectrophotometer (Shimadzu Corporation, Kyoto, Japan, website: www.shimadzu.com). TDSL samples of 60.1 OD were irradiated with 295 nm light, to selectively excite tryptophan residues of the protein and emission spectra were recorded above 300 nm. In fluorescence quenching experiments, small aliquots of 5 M quencher stocks were added to protein samples and fluorescence spectra were recorded after each addition. The iodide stock solution contained 0.2 mM sodium thiosulfate to prevent the formation of triiodide ðI 3 Þ. Fluorescence intensities were corrected for volume changes before further analysis of the quenching data. All measurements were performed at 25 °C. All quenching experiments were carried out in duplicate, which showed high reproducibility and the average results are reported. 2.4. Time-resolved fluorescence measurements
2.1. Materials Trichosanthes dioica seeds were obtained from the University of Agricultural Science, Rajendra Nagar (Hyderabad, India). Potassium iodide was obtained from Qualigens (Mumbai, India). Cesium chloride, acrylamide, succinimide, lactose, methyl-b-D-galactopyranoside and guanidine hydrochloride were obtained from Sigma (St. Louis, MO, USA). All other chemical used were of the highest available quality. 2.2. Trichosanthes dioica seed lectin TDSL was purified by affinity chromatography on cross-linked guar gum as described earlier [4]. Purity and hemagglutination activity of the affinity-eluted protein were consistent with earlier observations [4]. For quenching studies with denatured TDSL, the protein was incubated with 6 M Gdn.Cl overnight at room temperature. For experiments with sugar-bound TDSL, the lectin samples and the quencher stocks were made 0.1 M in the sugar (MebGal or lactose). All solutions were made in 10 mM sodium phosphate containing 150 mM NaCl and 0.02% sodium azide (PBS). 2.3. Steady-state fluorescence spectroscopy All emission spectra were recorded on a Spex Fluoromax-3 fluorescence spectrometer from Jobin-Yvon (Edi-
Fluorescence lifetime measurements were performed using an IBH-5000 single photon counting spectrofluorimeter from HORIBA Jobin Yvon IBH Ltd. (Glasgow, United Kingdom; website: www.ibh.co.uk). A hydrogen flash lamp of pulse width of 1.4 ns was used for excitation and a Hamamatasu photomultiplier 3235 was used to detect the fluorescence. Samples were excited at 295 nm and emission intensities were recorded at the kmax of the respective emission spectrum (native TDSL: 328 nm and denatured TDSL: 343 nm). Slit widths of 16 nm were used on both the excitation and emission monochromators. The resultant decay curves were analyzed by a multiexponential iterative fitting program supplied by IBH.
3. Results 3.1. Quenching of the intrinsic fluorescence emission of TDSL Quenching studies were carried out with the T. dioica seed lectin using two neutral quenchers (acrylamide and succinimide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The fluorescence emission spectra of native TDSL and TDSL denatured with 6 M Gdn.Cl, recorded in the absence and in the presence of increasing concentrations of acrylamide are shown in
N.A.M. Sultan, M.J. Swamy / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 93–100
Fig. 1(A) and (B), respectively. In these figures spectrum 1 corresponds to the lectin alone, while the spectra numbered 2–18 correspond to those recorded in the presence of increasing concentrations of the quencher. A comparison of the spectra shows that the emission kmax of the native lectin seen at 328 nm (Fig. 1(A), spectrum 1) is red-shifted to about 343 nm upon denaturation (Fig. 1(B), spectrum 1). Further, the spectra also show that the extent of quenching by acrylamide is higher in the presence of Gdn.Cl, clearly indicating that accessibility of the fluorophores to the quencher increased upon unfolding of the polypeptide chains of the lectin. Fluorescence intensity at kmax also decreased by about 44% upon denaturation (not shown). Since the emission maxima and relative intensities of tryptophan emission spectra in non-polar and polar solvents are similar to those of native and denatured TDSL, respectively, these observations suggest that, in native TDSL, the tryptophan residues are buried in the hydrophobic core of the folded polypeptide chains and upon unfolding of the protein, these residues become exposed to the bulk aqueous medium (cf. [12–14]). This conclusion is strongly supported by the fact that, in fluorescence quenching experiments, the extent of quenching with all quenchers increases upon denaturation of the lectin (see below). Further, this interpretation is also consistent with the inability of N-bromosuccinimide to oxidize the indole side chains of the tryptophan residues of TDSL in the native condition, whereas a maximum of two Trp residues per dimer could be oxidized by NBS upon denaturation of the protein [4]. However, it is likely that some residual structure is still present in the protein structure even after incubation with 6 M Gdn.Cl
(B)
Fluorescence Intensity (Arb. units)
(A) 1
1
18
18
95
because tryptophan alone in buffer yielded a fluorescence spectrum with a kmax around 350 (±1) nm. This is most likely due to the presence of disulfide bonds in the protein which hold different parts of the polypeptide covalently connected, resulting in retention of some residual order in the protein structure. The extent of quenching observed in each case, at a resultant quencher concentration of 0.5 M, is shown in Table 1. Acrylamide was the most effective among the four quenchers used, quenching about 27.2% of the total fluorescence of the protein. The other neutral quencher, succinimide, which is somewhat bulkier, quenched about 24.0% of the fluorescence. The charged quenchers, I and Cs+, which can not access the tryptophan residues buried in the protein matrix, were found to quench only about 4.4% and 9.5%, respectively, of the total fluorescence intensity of TDSL. Presence of 0.1 M lactose did not noticeably affect the quenching by acrylamide but decreased the quenching by succinimide, I and Cs+ to 21.3%, 2.1% and 5.2%, respectively. Denaturation resulted in a considerable increase in the quenching by all quenchers and the extent of quenching observed was 72.7%, 46.6%, 49.7%, and 20.9%, with acrylamide, succinimide, iodide and cesium ions, respectively (Table 1). 3.2. Analysis of the steady-state fluorescence quenching The steady-state fluorescence quenching data where analyzed using Stern–Volmer and modified Stern– Volmer equations (Eqs. (1) and (2), respectively) [15]: F 0 =F ¼ 1 þ K SV ½Q
ð1Þ
F 0 =DF ¼ fa1 þ 1=ðK a fa ½QÞ
ð2Þ
where F0 and F are the relative fluorescence intensities in the absence and presence of the quencher, respectively, [Q] is the quencher concentration, KSV is Stern–Volmer quenching constant, DF = (F0 F) is the change in fluorescence intensity at any point in the quenching titration, fa is the fraction of the total fluorophores accessible to the quencher and Ka is the corresponding Stern–Volmer constant for the accessible fraction of the fluorophores. Eq. (1) indicates that the slope of a plot of F0/F versus [Q] (Stern–Volmer plot) is equal to
Table 1 Extent of quenching of fluorescence of TDSL by different quenchers 310
330
350
370
310
330
350
370
390
Quencher
Quenching (%) Native
With 0.1 M lactose
In 6 M Gdn.HCl
Acrylamide Succinimide Iodide ion Cesium ion
27.2 24.0 4.4 9.5
27.4 21.3 2.1 5.2
72.7 46.6 49.7 20.9
Wavelength (nm)
Fig. 1. Fluorescence spectra of TDSL in the absence and in the presence of acrylamide. (A) Under native conditions; (B) in the presence of 6 M Gdn.HCl. Spectrum 1 corresponds to TDSL alone and spectra 2–18 correspond to increasing concentrations of acrylamide.
The final quencher concentration in each case was 0.5 M.
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2.0
(A)
2.6
(B)
(D)
1.8
5
2.2
1.3
1.8
1.2
1.4
1.1
1.0
1.0
1.6
Fo/F
4
3
1.4
2
1.2
1
1.0
0.00
1.4
(C)
0.25
0.50
0.75
0.0
0.2
0.4
0.6
0.0
0.3
0.6
0.9
0.0
0.3
0.6
0.9
[Quencher] (M)
Fig. 2. Stern–Volmer plots of fluorescence quenching for TDSL. The different quenchers used are (A) acrylamide, (B) succinimide, (C) iodide ion, and (D) cesium ion. (s) Native protein, (j) in the presence 0.1 M lactose, and (m) denatured in 6 M Gdn.HCl.
KSV, whereas Eq. (2) shows that the slope of a plot of F0/ DF versus [Q]1 (modified Stern–Volmer plot) gives the value of (Kafa)1, and its Y-intercepts gives the value of fa1 . Stern–Volmer plots for the quenching of TDSL with different quenchers are shown in Fig. 2. With native TDSL for all the quenchers studied, these plots exhibit biphasic patterns, demonstrating the heterogeneity in the accessibility of the various quenchers to the tryptophan residues of the lectin. From the slopes of the two phases of these plots the corresponding Stern–Volmer constants (KSV1 and KSV2) were obtained and listed in Table 2. The modified Stern–Volmer plots obtained with all the four quenchers are shown in Fig. 3. Y-intercepts
and slopes of these plots were used to obtain fa and Ka. It can be seen from the data shown in this table that in native TDSL about 66% of the fluorescence is accessible to acrylamide, whereas succinimide could access only 37% of the total fluorescence intensity. Iodide ion and cesium ion could access only 8% and 12%, respectively, of the total fluorescence intensity. Denaturation with 6 M Gdn.Cl led to 100% accessibility for acrylamide, whereas for succinimide it increased to 91%. For the ionic quenchers, I and Cs+, the fraction accessible increased to 77% and 41%, respectively. The Stern–Volmer plots are all either linear or downward curving (Fig. 2), a behavior expected from dynamic fluorescence quenching of proteins with more than one tryptophan residues in different accessible
Table 2 Summary of parameters obtained from the intrinsic fluorescence quenching with different quenchers and from time-resolved fluorescence measurements Quencher and condition
KSV1 (M1)
109 kq1 (M1 s1)
KSV2 (M1)
109 kq2 (M1 s1)
fa (%)
Ka (M1)
Acrylamide Native With 0.1 M lactose In 6 M Gdn.HCl
0.869 0.899 5.580
0.323 0.339 2.068
0.635 0.649
0.236 0.245
0.66 0.64 1.00
1.458 1.583 5.180
Succinimide Native With 0.1 M lactose In 6 M Gdn.HCl
0.684 0.525 1.902
0.255 0.198 0.705
0.517
0.192
1.381
0.512
0.37 0.43 0.91
3.276 1.738 2.176
Iodide ion (I ) Native With 0.1 M lactose In 6 M Gdn.HCl
0.191 0.034 2.292
0.071 0.013 0.850
0.029
0.011
1.714
0.635
0.08 0.04 0.77
3.243 2.340 3.476
Cesium ion (Cs+) Native With 0.1 M lactose In 6 M Gdn.HCl
0.486 0.106 0.650
0.181 0.040 0.241
0.119
0.044
0.338
0.125
0.12 0.14 0.41
6.536 1.215 2.163
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25
(A)
(B)
160
(C)
97
(D)
160 20
20 120 120 15
Fo/∆F
15
80 80
10
10
5
5
0 5
10
15
20
0
0
0 0
40
40
0
5
10
15
20
0 -1
5
10
15
20
0
5
10
15
20
-1
[Quencher] (M )
Fig. 3. Modified Stern–Volmer plots for the quenching of the intrinsic fluorescence of TDSL with different quenchers. (A) Acrylamide, (B) succinimide, (C) iodide ion, and (D) cesium ion. (s) Native TDSL, (j) TDSL in the presence of 0.1 M lactose and (m) TDSL denatured with 6 M Gdn.HCl.
environments. For dynamic quenching, KSV is equal to the product s0 Æ kq (or kq = KSV/s0), where s0 is the exited state lifetime of the fluorophore in the absence of quencher and kq is the bimolecular rate constant for the elementary interaction between the fluorophore excited state and the quencher. For systems showing multiexponential fluorescence decay curves, s0 is the average of all individual lifetimes (see Section 3.3). From the values of KSV and s0 for each quencher, the kq values were calculated and listed in Table 2. 3.3. Fluorescence decay and excited state lifetimes Fig. 4 shows fluorescence decay curve of the native TDSL. Similar curves were also obtained for denatured 3.5 Lamp profile Decay profile non-linear fit
3.0
i
-3
i
where i = 1, 2 and ai is the weighing factor of si. In all cases it is seen that the longer s has the smaller a (for example, a1 = 0.137 and a2 = 0.042, for native TDSL). The ai and si values obtained for TDSL in the native state, in the presence of 0.1 M lactose and upon denaturation are listed in Table 3.
2.5
10 X Counts
TDSL and sugar-bound TDSL. In all cases, the decay curves could be best fitted to a biexponential decay function (v2 6 1.12), i.e., two different lifetimes of the exited tryptophan residues could be observed. While monoexponential fits yielded significantly larger errors (v2 P 2.0), triexponential fits did not noticeably improve the quality of the fits. For native TDSL the two lifetimes obtained from this analysis are 1.53 and 4.09 ns (v2 = 1.12), whereas in the presence of 0.1 M lactose the longer lifetime decreased to 3.4 ns whereas the shorter lifetime increased slightly to 1.65 ns. Upon denaturation both the lifetimes decreased to 3.5 and 1.4 ns, respectively. From the above data, the average lifetime of fluorescence decay, s0, was calculated according to the equation [16]: , X X 2 s0 ¼ ai si ai s i ð3Þ
2.0 1.5 1.0 0.5
Table 3 Lifetimes of TDSL fluorescence and their weighing factors obtained from fitting of fluorescence decay curves to a biexponential function
0.0 3
4
5
6
7
Time (ns) Fig. 4. Time-resolved fluorescence decay profile of native TDSL: () lamp profile and () decay profile. The solid line is the best least square fit of the decay profile to a biexponential function (v2 = 1.1).
Sample
a1
s1
a2
s2
s0
v2
Native TDSL Denatured TDSL TDSL in 0.1 M lactose
0.042 0.068 0.034
4.09 3.50 3.40
0.137 0.105 0.129
1.53 1.40 1.65
2.69 2.70 2.65
1.12 1.01 1.11
The fitting error is shown (v2).
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4. Discussion The tryptophan exposure and environment in proteins can be investigated by quenching its fluorescence employing small molecule quenchers, such as acrylamide and iodide ion, which is also referred to as solute perturbation technique. Quenching by these species proceeds mainly through physical interaction between the fluorophore and the quencher; therefore, it is directly dependent on the extent to which the quencher can access the fluorophore in the protein. While charged quenchers such as I and Cs+ can quench only the surface exposed tryptophan residues, neutral quenchers such as acrylamide and succinimide can penetrate into the interior of the protein matrix and can quench partially buried tryptophan residues also. Because the depth of penetration of the latter two quenchers depends on their molecular radii, with the smaller acrylamide being able to penetrate deeper into the protein interior and thus quench the fluorescence to a larger extent, a comparison of the relative quenching efficiencies of these two quenchers provides a measure of the degree of burial of the tryptophan residues of the protein. Thus, by employing all the four quenchers mentioned above, considerable insights can be derived on the environment and exposure of tryptophan residues in proteins. In this study, all the above four quenchers (acrylamide, succinimide, I, and Cs+) were used to study the tryptophan residues of native TDSL in the absence and in the presence of sugars as well as upon denaturation. Previous chemical modification studies have suggested that there are 2.0 tryptophan residues/protein dimer. The emission maximum of native TDSL, which is seen at 328 nm (Fig. 1), strongly suggests that both these tryptophan residues are in a predominantly hydrophobic environment, i.e., they are buried in the hydrophobic interior of the protein. Sugar binding does not lead to any detectable change in the emission kmax and the changes induced in the emission intensity by the binding of lactose and Meb Gal was rather small, indicating that the environment of the tryptophan residues is not significantly altered by saccharide binding. The large red shift in the emission maximum of the protein upon denaturation by 6 M Gdn.Cl (Fig. 1) indicates greater exposure of the tryptophan residues to the aqueous environment. This is also evident from the increase in the extent of quenching observed with different quenchers in the presence of 6 M Gdn.Cl (see Table 1). Similar observations were made on SGSL and TCSL [17,18]. Because the tryptophan residues of TDSL appear to be buried in the protein matrix, the extent of quenching observed with different quenchers and the degree of accessibility of the fluorophores to them vary depending on the size and charge of the quencher (Table 1). Although both acrylamide and succinimide are neutral,
due to its larger size the approach of succinimide to the buried fluorophores is expected to be more restricted [19]. Therefore, the decrease in the extent of quenching and the accessible fraction of fluorophores for succinimide when compared to acrylamide, suggests that the tryptophan residues of TDSL are at least partially buried in the protein interior. The ionic quenchers, I and Cs+, being charged, can not diffuse, unlike the neutral quenchers, within the hydrophobic core of proteins and thus do not probe buried tryptophan residues. This makes them especially useful to study the environment of surface or exposed tryptophan residues [15,17]. For native TDSL, as can be seen from Tables 1 and 2, fluorescence quenching by these quenchers is substantially small and shows small quenching parameters. On the other hand, for denatured lectin, where the polypeptide chain is unfolded and tryptophan residues are exposed to the bulk solvent, the fluorescence quenching parameters obtained with these ionic quenchers increase significantly. This clearly gives further support to the conclusion drawn from an analysis of the emission spectra (see Section 3.1), that the tryptophan residues of native TDSL are buried in its hydrophobic core. Two more comments can be made here about the differences in quenching parameters between native and denatured lectin. Firstly, the increase in fluorescence quenching parameters of TDSL with Cs+, upon denaturation of the lectin, is not as large as those observed with other quenchers, especially with I (see Table 2). Based on this observation, one can infer the presence of at least one positively charged amino acid residue, under the experimental conditions, close to one or more tryptophan residues. Similar observations were reported for another seed lectin from Cucurbitaceae, TCSL [18]. Secondly, only for acrylamide, denaturation of TDSL results in complete accessibility of the quencher to the fluorophores (fa value equal to 1.0, linear dependence of F0/F on [Q], and nearly equal values of KSV and Ka). This most likely results from the neutral character and high efficiency of acrylamide as a protein fluorescence quencher. The inability of succinimide to achieve 100% accessibility suggests the presence of some local order in the protein polypeptide chain even in the presence of 6 M Gdn.Cl, which allows only partial accessibility to most likely one Trp residue. As mentioned in Section 3.1, this residual order in the protein structure is most likely due to the presence of disulfide bonds in the protein. Since acrylamide could achieve 100% accessibility, this local order in the protein structure does not prevent it from getting close to the Trp residue that is partially shielded from succinimide. Lactose binding to TDSL leads to a significant decrease of its fluorescence quenching parameters with all quenchers except acrylamide. This effect of sugar binding on the fluorescence quenching of TDSL
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suggests that the bound sugar makes it harder to the bulky quencher, succinimide as well as to the ionic quenchers (I and Cs+) to diffuse into the hydrophobic core of the lectin. The reason why quenching by acrylamide is not affected by bound sugar could be again the neutrality (compared to I and Cs+), size and efficiency (compared to succinimide) of this quencher. Sugar binding to two other Cucurbitaceae lectins, SGSL [17] and TCSL [18], did not significantly alter the fluorescence quenching by external quenchers. However, for another Cucurbitaceae seed lectin, Momordica charantia lectin, MCL, various quenchers showed decrease in fluorescence quenching parameters upon binding of a specific sugar, lactose [20]. These different effects of sugar binding by these similar lectins on their fluorescence quenching behavior can be explained in view of the amino acid residues involved in their sugar binding activity. In MCL, chemical modification studies show that both tryptophan and tyrosine are involved in the sugar binding activity [21]. Therefore, the presence of a specific sugar in the quenching medium is likely to provide some protection to the fluorescent Trp residues against the quenchers leading to smaller quenching parameters. The same can be said for TDSL, in which tyrosine, whose excitation energy usually is transferred to tryptophan, is involved in the lectin activity [4]. On the other hand, in both SGSL and TCSL, neither tryptophan nor tyrosine are involved in the sugar binding [17,22,23]. Therefore, in the absence of significant ligand-induced conformational changes, which can lead to differences in the accessibility of the Trp residues, for these two proteins the fluorescence quenching is not expected to be altered by the presence a specific sugar, which was experimentally observed [17,18]. Time-resolved fluorescence studies showed the presence of two components in the fluorescence decay profiles of TDSL. There are at least two reasons for multiplicity of tryptophan lifetime in proteins: (a) the presence of tryptophan in several different rotamers that are quenched to different extents by the functional groups (peptide backbone and amino acid side chains) in the microenvironment surrounding the indole ring [24] (for this reason, even single-tryptophan proteins exhibit multiexponential fluorescence decay), and (b) the heterogeneity of the environments of various tryptophan populations in multitryptophan proteins, such as TDSL in this study. In summary, the fluorescence quenching studies reported here demonstrate that the tryptophan residues of TDSL are in a heterogeneous hydrophobic environment. Lactose, occupying the sugar binding site, hinders the accessibility of all quenchers (except acrylamide) to the interior of the TDSL. Fluorescence quenching parameters obtained with Cs+ (compared to the other quenchers, especially I) suggests the presence of one
99
or more positively charged amino acid close to some of the tryptophan residues.
5. Abbreviations TDSL SGSL TCSL MCL PBS
Trichosanthes dioica seed lectin snake gourd (Trichosanthes anguina) seed lectin Trichosanthes cucumerina seed lectin Momordica charantia lectin 10 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride and 0.02% sodium azide Gdn.HCl guanidinium chloride (guanidine hydrochloride) bME b-mercaptoethanol MebGal methyl-b-D-galactopyranoside Trp tryptophan
Acknowledgements This work was supported by a research project from the Department of Science and Technology (India) to MJS. NAMS was supported by a Research Fellowship from the Sanaa´ University, Yemen. The IBH 5000 time-resolved fluorescence spectrometer was supported by the DST (India). We thank Professor A. Samanta for generous access to the timeresolved fluorescence spectrometer and Mr. Sandeep Banthia and Mr. Rana Karmakar for help with measurements on it.
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[8] J.R. Lakowicz, Principles of Fluorescence Spectroscopy, second ed., Kluwer Academic Publishers, 1999. [9] M.R. Eftink, C.A. Ghiron, Fluorescence quenching studies with proteins, Anal. Biochem. 114 (1981) 199–227. [10] M.K. Das, M.I. Khan, A. Surolia, Fluorimetric studies of the binding of Momordica charantia (bitter gourd) lectin with ligands, Biochem. J. 195 (1981) 341–343. [11] M. Kakitani, B. Tonomura, K. Hiromi, Fluorometric study on the interaction of amino acids and ATP with valyl-tRNA synthetase from Bacillus stearothermophilus, J. Biochem. 101 (1987) 477–484. [12] E.A. Burstein, N.S. Vedenkina, M.N. Ivkova, Fluorescence and the location of tryptophan residues in protein molecules, Photochem. Photobiol. 18 (1973) 263–279. [13] M.R. Eftink, C.A. Ghiron, Exposure of tryptophanyl residues in proteins. Quantitative determination by fluorescence quenching studies, Biochemistry 15 (1976) 672–680. [14] S.A. Barghouthi, J. Perrault, L.H. Holmes, Effect of solvents on the fluorescence emission spectra of 1-anilino-8-naphthalene sulfonic acid: a physical chemistry experiment, Chem. Educat. 3 (1998) 1–10. [15] S.S. Lehrer, Solute perturbation of protein fluorescence. The quenching of tryptophyl fluorescence of model compounds and of lysozyme by iodide ion, Biochemistry 10 (1971) 3254–3263. [16] A. Grinvald, I.Z. Steinberg, On the analysis of the fluorescence decay kinetics by the method of least squares, Anal. Biochem. 59 (1974) 583–598.
[17] S.S. Komath, M.J. Swamy, Fluorescence quenching, timeresolved fluorescence and chemical modification studies on snake gourd (Trichosanthes anguina) seed lectin, J. Photochem. Photobiol. B: Biol. 50 (1999) 109–118. [18] R. Kenoth, M.J. Swamy, Steady-state and time-resolved fluorescence studies on Trichosanthes cucumerina seed lectin, J. Photochem. Photobiol. B: Biol. 69 (2003) 193–201. [19] M.R. Eftink, C.A. Ghiron, Indole fluorescence quenching on proteins and model system: Use of the inefficient quencher succinimide, Biochemistry 23 (1984) 3891–3899. [20] T. Mazumdar, N. Gaur, A. Surolia, The physicochemical properties of the galactose-specific lectin from Momordica charantia, Eur. J. Biochem. 113 (1981) 463–470. [21] P. Padma, S.S. Komath, M.J. Swamy, Fluorescence quenching and time-resolved fluorescence studies on Momordica charantia (bitter gourd) seed lectin, Biochem. Mol. Biol. Int. 45 (1998) 911– 920. [22] S.S. Komath, S.K. Nadimpalli, M.J. Swamy, Identification of histidine residues in the sugar binding site of snake gourd (Trichosanthes anguina) seed lectin, Biochem. Mol. Biol. Int. 44 (1998) 107–117. [23] R. Kenoth, P. Padma, S. Sarada, M.J. Swamy, Role of histidine residues in sugar-binding activity of Trichosanthes cucumerina seed lectin, J. Biochem. Mol. Biol. Biophys. 4 (2000) 423–431. [24] A.G. Szabo, D.M. Rayner, Fluorescence decay of tryptophan conformers in aqueous solution, J. Am. Chem. Soc. 102 (1980) 554–563.
Fluorescence quenching and time-resolved fluorescence studies on Trichosanthes dioica seed lectin Nabil Ali Mohammed Sultan, Musti J. Swamy
*
School of Chemistry, University of Hyderabad, Hyderabad 500 046, India Received 21 January 2005; received in revised form 10 March 2005; accepted 10 March 2005 Available online 29 April 2005
Abstract Fluorescence quenching and time-resolved fluorescence studies have been carried out on the Trichosanthes dioica seed lectin (TDSL). The emission kmax of native TDSL, seen at 328 nm, shifts to 343 nm upon denaturation with 6 M guanidinium chloride. Quenching titrations were performed with neutral (acrylamide and succinimide) and ionic (I and Cs+) quenchers in order to probe the exposure and accessibility of tryptophan residues of the protein. Maximum quenching was observed with acrylamide, followed by succinimide, iodide and Cs+. Dramatic increase in the extent of quenching and other quenching parameters by all the quenchers were observed upon denaturation of TDSL, suggesting that all the tryptophan residues in native TDSL are buried in the hydrophobic core of the protein. Increase in the extent of quenching upon denaturation of TDSL was maximum with I and minimum with Cs+, suggesting the presence of positively charged residue(s), near at least one tryptophan residue. Addition of saccharide ligands such as methyl-b-D-galactopyranoside and lactose led to a small, but reproducible decrease in the fluorescence intensity of the lectin. The presence of lactose provided a partial protection against quenching by I, Cs+ and succinimide, but not acrylamide. In timeresolved fluorescence measurements the fluorescence decay curves could be best fitted to biexponential patterns with lifetimes of 4.09 and 1.53 ns for native lectin, 3.40 and 1.65 ns for the lectin in presence of 0.1 M lactose and 3.50 and 1.40 ns for denatured lectin. Ó 2005 Elsevier B.V. All rights reserved. Keywords: Intrinsic fluorescence; Agglutinin; Tryptophan; Acrylamide; Succinimide; Iodide ion; Cesium ion
1. Introduction Plant lectins are proteins with at least one non-catalytic domain which binds reversibly and specifically a mono- or oligosaccharide [1]. Lectins have elicited considerable interest due to a variety of interesting biological properties exhibited by them such as blood-group specific hemagglutination, mitogenicity, and the ability to distinguish between normal and malignant cells. In some plants, lectins may function as defense proteins whereas in others they may be responsible for the symbiosis between plant roots and nodulating Rhizobia. In * Corresponding author. Tel.: +91 40 2301 1071; fax: +91 40 2301 2460. E-mail address: [email protected] (M.J. Swamy). URL: http://202.41.85.161/~mjs/ (M.J. Swamy).
1011-1344/$ - see front matter Ó 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jphotobiol.2005.03.003
addition, lectins are also widely used in the detection, isolation and characterization of glycoconjugates and in the fractionation of cells for their use in bone marrow transplantation [2,3]. All these interesting properties, functions and applications are manifested through the specific recognition of carbohydrates by lectins. Trichosanthes dioica seed lectin (TDSL) is a galactose/ N-acetylgalactosamine-specific, heterodimeric glycoprotein with at least one intersubunit disulfide bridge and an apparent molecular mass of 55 kDa [4]. It exhibits a higher preference for b-galactosides over the corresponding a-anomeric derivatives. Chemical modification studies have implicated tyrosine residues in its carbohydrate binding activity. Circular dichroism spectroscopic studies indicate that its secondary structure is made of 10% a-helix, 37% b-sheet, 21% turns and the rest
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unordered chain [4]. TDSL is similar to several other seed lectins from Cucurbitaceae such as Trichosanthes kirilowii lectin, snake gourd (Trichosanthes anguina) seed lectin, and Trichosanthes cucumerina seed lectin, purified in this laboratory and elsewhere [5–7]. The intrinsic fluorescence of proteins arises primarily from the side chains of tyrosine and tryptophan residues. By exciting the protein sample at 295 nm or above, where tyrosine residues do not absorb, it is possible to study the fluorescence due to tryptophan alone [8]. Changes in the fluorescence characteristics of tryptophan residues are used widely to obtain information about conformational transitions in proteins, association of subunits in oligomeric proteins, protein unfolding or ligand binding to proteins [8–11]. The indole side chains of the tryptophan residues of TDSL could not be modified by N-bromosuccinimide under native condition and therefore appear to be buried in the protein matrix [4]. In order to investigate in greater detail the degree of exposure and environment of the tryptophan residues in TDSL, we carried out fluorescence quenching and time-resolved fluorescence studies on this lectin under native and denaturing conditions. The results obtained are presented here. 2. Materials and method
son, NJ, USA, website: http://www.jobinyvon.com). Slit widths of 3 and 6 nm were used on the excitation and emission monochromators, respectively. The integration time was set at 0.3 s. The calibration of the spectrometer was checked frequently using the Raman scattering band for water. The mcorrect feature in the operation software was routinely used during the spectral scan to correct for the photomultiplier characteristics. Absorption spectral measurements were made using a Shimadzu UV3101PC double-beam spectrophotometer (Shimadzu Corporation, Kyoto, Japan, website: www.shimadzu.com). TDSL samples of 60.1 OD were irradiated with 295 nm light, to selectively excite tryptophan residues of the protein and emission spectra were recorded above 300 nm. In fluorescence quenching experiments, small aliquots of 5 M quencher stocks were added to protein samples and fluorescence spectra were recorded after each addition. The iodide stock solution contained 0.2 mM sodium thiosulfate to prevent the formation of triiodide ðI 3 Þ. Fluorescence intensities were corrected for volume changes before further analysis of the quenching data. All measurements were performed at 25 °C. All quenching experiments were carried out in duplicate, which showed high reproducibility and the average results are reported. 2.4. Time-resolved fluorescence measurements
2.1. Materials Trichosanthes dioica seeds were obtained from the University of Agricultural Science, Rajendra Nagar (Hyderabad, India). Potassium iodide was obtained from Qualigens (Mumbai, India). Cesium chloride, acrylamide, succinimide, lactose, methyl-b-D-galactopyranoside and guanidine hydrochloride were obtained from Sigma (St. Louis, MO, USA). All other chemical used were of the highest available quality. 2.2. Trichosanthes dioica seed lectin TDSL was purified by affinity chromatography on cross-linked guar gum as described earlier [4]. Purity and hemagglutination activity of the affinity-eluted protein were consistent with earlier observations [4]. For quenching studies with denatured TDSL, the protein was incubated with 6 M Gdn.Cl overnight at room temperature. For experiments with sugar-bound TDSL, the lectin samples and the quencher stocks were made 0.1 M in the sugar (MebGal or lactose). All solutions were made in 10 mM sodium phosphate containing 150 mM NaCl and 0.02% sodium azide (PBS). 2.3. Steady-state fluorescence spectroscopy All emission spectra were recorded on a Spex Fluoromax-3 fluorescence spectrometer from Jobin-Yvon (Edi-
Fluorescence lifetime measurements were performed using an IBH-5000 single photon counting spectrofluorimeter from HORIBA Jobin Yvon IBH Ltd. (Glasgow, United Kingdom; website: www.ibh.co.uk). A hydrogen flash lamp of pulse width of 1.4 ns was used for excitation and a Hamamatasu photomultiplier 3235 was used to detect the fluorescence. Samples were excited at 295 nm and emission intensities were recorded at the kmax of the respective emission spectrum (native TDSL: 328 nm and denatured TDSL: 343 nm). Slit widths of 16 nm were used on both the excitation and emission monochromators. The resultant decay curves were analyzed by a multiexponential iterative fitting program supplied by IBH.
3. Results 3.1. Quenching of the intrinsic fluorescence emission of TDSL Quenching studies were carried out with the T. dioica seed lectin using two neutral quenchers (acrylamide and succinimide), an anionic quencher (iodide ion) and a cationic quencher (cesium ion). The fluorescence emission spectra of native TDSL and TDSL denatured with 6 M Gdn.Cl, recorded in the absence and in the presence of increasing concentrations of acrylamide are shown in
N.A.M. Sultan, M.J. Swamy / Journal of Photochemistry and Photobiology B: Biology 80 (2005) 93–100
Fig. 1(A) and (B), respectively. In these figures spectrum 1 corresponds to the lectin alone, while the spectra numbered 2–18 correspond to those recorded in the presence of increasing concentrations of the quencher. A comparison of the spectra shows that the emission kmax of the native lectin seen at 328 nm (Fig. 1(A), spectrum 1) is red-shifted to about 343 nm upon denaturation (Fig. 1(B), spectrum 1). Further, the spectra also show that the extent of quenching by acrylamide is higher in the presence of Gdn.Cl, clearly indicating that accessibility of the fluorophores to the quencher increased upon unfolding of the polypeptide chains of the lectin. Fluorescence intensity at kmax also decreased by about 44% upon denaturation (not shown). Since the emission maxima and relative intensities of tryptophan emission spectra in non-polar and polar solvents are similar to those of native and denatured TDSL, respectively, these observations suggest that, in native TDSL, the tryptophan residues are buried in the hydrophobic core of the folded polypeptide chains and upon unfolding of the protein, these residues become exposed to the bulk aqueous medium (cf. [12–14]). This conclusion is strongly supported by the fact that, in fluorescence quenching experiments, the extent of quenching with all quenchers increases upon denaturation of the lectin (see below). Further, this interpretation is also consistent with the inability of N-bromosuccinimide to oxidize the indole side chains of the tryptophan residues of TDSL in the native condition, whereas a maximum of two Trp residues per dimer could be oxidized by NBS upon denaturation of the protein [4]. However, it is likely that some residual structure is still present in the protein structure even after incubation with 6 M Gdn.Cl
(B)
Fluorescence Intensity (Arb. units)
(A) 1
1
18
18
95
because tryptophan alone in buffer yielded a fluorescence spectrum with a kmax around 350 (±1) nm. This is most likely due to the presence of disulfide bonds in the protein which hold different parts of the polypeptide covalently connected, resulting in retention of some residual order in the protein structure. The extent of quenching observed in each case, at a resultant quencher concentration of 0.5 M, is shown in Table 1. Acrylamide was the most effective among the four quenchers used, quenching about 27.2% of the total fluorescence of the protein. The other neutral quencher, succinimide, which is somewhat bulkier, quenched about 24.0% of the fluorescence. The charged quenchers, I and Cs+, which can not access the tryptophan residues buried in the protein matrix, were found to quench only about 4.4% and 9.5%, respectively, of the total fluorescence intensity of TDSL. Presence of 0.1 M lactose did not noticeably affect the quenching by acrylamide but decreased the quenching by succinimide, I and Cs+ to 21.3%, 2.1% and 5.2%, respectively. Denaturation resulted in a considerable increase in the quenching by all quenchers and the extent of quenching observed was 72.7%, 46.6%, 49.7%, and 20.9%, with acrylamide, succinimide, iodide and cesium ions, respectively (Table 1). 3.2. Analysis of the steady-state fluorescence quenching The steady-state fluorescence quenching data where analyzed using Stern–Volmer and modified Stern– Volmer equations (Eqs. (1) and (2), respectively) [15]: F 0 =F ¼ 1 þ K SV ½Q
ð1Þ
F 0 =DF ¼ fa1 þ 1=ðK a fa ½QÞ
ð2Þ
where F0 and F are the relative fluorescence intensities in the absence and presence of the quencher, respectively, [Q] is the quencher concentration, KSV is Stern–Volmer quenching constant, DF = (F0 F) is the change in fluorescence intensity at any point in the quenching titration, fa is the fraction of the total fluorophores accessible to the quencher and Ka is the corresponding Stern–Volmer constant for the accessible fraction of the fluorophores. Eq. (1) indicates that the slope of a plot of F0/F versus [Q] (Stern–Volmer plot) is equal to
Table 1 Extent of quenching of fluorescence of TDSL by different quenchers 310
330
350
370
310
330
350
370
390
Quencher
Quenching (%) Native
With 0.1 M lactose
In 6 M Gdn.HCl
Acrylamide Succinimide Iodide ion Cesium ion
27.2 24.0 4.4 9.5
27.4 21.3 2.1 5.2
72.7 46.6 49.7 20.9
Wavelength (nm)
Fig. 1. Fluorescence spectra of TDSL in the absence and in the presence of acrylamide. (A) Under native conditions; (B) in the presence of 6 M Gdn.HCl. Spectrum 1 corresponds to TDSL alone and spectra 2–18 correspond to increasing concentrations of acrylamide.
The final quencher concentration in each case was 0.5 M.
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2.0
(A)
2.6
(B)
(D)
1.8
5
2.2
1.3
1.8
1.2
1.4
1.1
1.0
1.0
1.6
Fo/F
4
3
1.4
2
1.2
1
1.0
0.00
1.4
(C)
0.25
0.50
0.75
0.0
0.2
0.4
0.6
0.0
0.3
0.6
0.9
0.0
0.3
0.6
0.9
[Quencher] (M)
Fig. 2. Stern–Volmer plots of fluorescence quenching for TDSL. The different quenchers used are (A) acrylamide, (B) succinimide, (C) iodide ion, and (D) cesium ion. (s) Native protein, (j) in the presence 0.1 M lactose, and (m) denatured in 6 M Gdn.HCl.
KSV, whereas Eq. (2) shows that the slope of a plot of F0/ DF versus [Q]1 (modified Stern–Volmer plot) gives the value of (Kafa)1, and its Y-intercepts gives the value of fa1 . Stern–Volmer plots for the quenching of TDSL with different quenchers are shown in Fig. 2. With native TDSL for all the quenchers studied, these plots exhibit biphasic patterns, demonstrating the heterogeneity in the accessibility of the various quenchers to the tryptophan residues of the lectin. From the slopes of the two phases of these plots the corresponding Stern–Volmer constants (KSV1 and KSV2) were obtained and listed in Table 2. The modified Stern–Volmer plots obtained with all the four quenchers are shown in Fig. 3. Y-intercepts
and slopes of these plots were used to obtain fa and Ka. It can be seen from the data shown in this table that in native TDSL about 66% of the fluorescence is accessible to acrylamide, whereas succinimide could access only 37% of the total fluorescence intensity. Iodide ion and cesium ion could access only 8% and 12%, respectively, of the total fluorescence intensity. Denaturation with 6 M Gdn.Cl led to 100% accessibility for acrylamide, whereas for succinimide it increased to 91%. For the ionic quenchers, I and Cs+, the fraction accessible increased to 77% and 41%, respectively. The Stern–Volmer plots are all either linear or downward curving (Fig. 2), a behavior expected from dynamic fluorescence quenching of proteins with more than one tryptophan residues in different accessible
Table 2 Summary of parameters obtained from the intrinsic fluorescence quenching with different quenchers and from time-resolved fluorescence measurements Quencher and condition
KSV1 (M1)
109 kq1 (M1 s1)
KSV2 (M1)
109 kq2 (M1 s1)
fa (%)
Ka (M1)
Acrylamide Native With 0.1 M lactose In 6 M Gdn.HCl
0.869 0.899 5.580
0.323 0.339 2.068
0.635 0.649
0.236 0.245
0.66 0.64 1.00
1.458 1.583 5.180
Succinimide Native With 0.1 M lactose In 6 M Gdn.HCl
0.684 0.525 1.902
0.255 0.198 0.705
0.517
0.192
1.381
0.512
0.37 0.43 0.91
3.276 1.738 2.176
Iodide ion (I ) Native With 0.1 M lactose In 6 M Gdn.HCl
0.191 0.034 2.292
0.071 0.013 0.850
0.029
0.011
1.714
0.635
0.08 0.04 0.77
3.243 2.340 3.476
Cesium ion (Cs+) Native With 0.1 M lactose In 6 M Gdn.HCl
0.486 0.106 0.650
0.181 0.040 0.241
0.119
0.044
0.338
0.125
0.12 0.14 0.41
6.536 1.215 2.163
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25
(A)
(B)
160
(C)
97
(D)
160 20
20 120 120 15
Fo/∆F
15
80 80
10
10
5
5
0 5
10
15
20
0
0
0 0
40
40
0
5
10
15
20
0 -1
5
10
15
20
0
5
10
15
20
-1
[Quencher] (M )
Fig. 3. Modified Stern–Volmer plots for the quenching of the intrinsic fluorescence of TDSL with different quenchers. (A) Acrylamide, (B) succinimide, (C) iodide ion, and (D) cesium ion. (s) Native TDSL, (j) TDSL in the presence of 0.1 M lactose and (m) TDSL denatured with 6 M Gdn.HCl.
environments. For dynamic quenching, KSV is equal to the product s0 Æ kq (or kq = KSV/s0), where s0 is the exited state lifetime of the fluorophore in the absence of quencher and kq is the bimolecular rate constant for the elementary interaction between the fluorophore excited state and the quencher. For systems showing multiexponential fluorescence decay curves, s0 is the average of all individual lifetimes (see Section 3.3). From the values of KSV and s0 for each quencher, the kq values were calculated and listed in Table 2. 3.3. Fluorescence decay and excited state lifetimes Fig. 4 shows fluorescence decay curve of the native TDSL. Similar curves were also obtained for denatured 3.5 Lamp profile Decay profile non-linear fit
3.0
i
-3
i
where i = 1, 2 and ai is the weighing factor of si. In all cases it is seen that the longer s has the smaller a (for example, a1 = 0.137 and a2 = 0.042, for native TDSL). The ai and si values obtained for TDSL in the native state, in the presence of 0.1 M lactose and upon denaturation are listed in Table 3.
2.5
10 X Counts
TDSL and sugar-bound TDSL. In all cases, the decay curves could be best fitted to a biexponential decay function (v2 6 1.12), i.e., two different lifetimes of the exited tryptophan residues could be observed. While monoexponential fits yielded significantly larger errors (v2 P 2.0), triexponential fits did not noticeably improve the quality of the fits. For native TDSL the two lifetimes obtained from this analysis are 1.53 and 4.09 ns (v2 = 1.12), whereas in the presence of 0.1 M lactose the longer lifetime decreased to 3.4 ns whereas the shorter lifetime increased slightly to 1.65 ns. Upon denaturation both the lifetimes decreased to 3.5 and 1.4 ns, respectively. From the above data, the average lifetime of fluorescence decay, s0, was calculated according to the equation [16]: , X X 2 s0 ¼ ai si ai s i ð3Þ
2.0 1.5 1.0 0.5
Table 3 Lifetimes of TDSL fluorescence and their weighing factors obtained from fitting of fluorescence decay curves to a biexponential function
0.0 3
4
5
6
7
Time (ns) Fig. 4. Time-resolved fluorescence decay profile of native TDSL: () lamp profile and () decay profile. The solid line is the best least square fit of the decay profile to a biexponential function (v2 = 1.1).
Sample
a1
s1
a2
s2
s0
v2
Native TDSL Denatured TDSL TDSL in 0.1 M lactose
0.042 0.068 0.034
4.09 3.50 3.40
0.137 0.105 0.129
1.53 1.40 1.65
2.69 2.70 2.65
1.12 1.01 1.11
The fitting error is shown (v2).
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4. Discussion The tryptophan exposure and environment in proteins can be investigated by quenching its fluorescence employing small molecule quenchers, such as acrylamide and iodide ion, which is also referred to as solute perturbation technique. Quenching by these species proceeds mainly through physical interaction between the fluorophore and the quencher; therefore, it is directly dependent on the extent to which the quencher can access the fluorophore in the protein. While charged quenchers such as I and Cs+ can quench only the surface exposed tryptophan residues, neutral quenchers such as acrylamide and succinimide can penetrate into the interior of the protein matrix and can quench partially buried tryptophan residues also. Because the depth of penetration of the latter two quenchers depends on their molecular radii, with the smaller acrylamide being able to penetrate deeper into the protein interior and thus quench the fluorescence to a larger extent, a comparison of the relative quenching efficiencies of these two quenchers provides a measure of the degree of burial of the tryptophan residues of the protein. Thus, by employing all the four quenchers mentioned above, considerable insights can be derived on the environment and exposure of tryptophan residues in proteins. In this study, all the above four quenchers (acrylamide, succinimide, I, and Cs+) were used to study the tryptophan residues of native TDSL in the absence and in the presence of sugars as well as upon denaturation. Previous chemical modification studies have suggested that there are 2.0 tryptophan residues/protein dimer. The emission maximum of native TDSL, which is seen at 328 nm (Fig. 1), strongly suggests that both these tryptophan residues are in a predominantly hydrophobic environment, i.e., they are buried in the hydrophobic interior of the protein. Sugar binding does not lead to any detectable change in the emission kmax and the changes induced in the emission intensity by the binding of lactose and Meb Gal was rather small, indicating that the environment of the tryptophan residues is not significantly altered by saccharide binding. The large red shift in the emission maximum of the protein upon denaturation by 6 M Gdn.Cl (Fig. 1) indicates greater exposure of the tryptophan residues to the aqueous environment. This is also evident from the increase in the extent of quenching observed with different quenchers in the presence of 6 M Gdn.Cl (see Table 1). Similar observations were made on SGSL and TCSL [17,18]. Because the tryptophan residues of TDSL appear to be buried in the protein matrix, the extent of quenching observed with different quenchers and the degree of accessibility of the fluorophores to them vary depending on the size and charge of the quencher (Table 1). Although both acrylamide and succinimide are neutral,
due to its larger size the approach of succinimide to the buried fluorophores is expected to be more restricted [19]. Therefore, the decrease in the extent of quenching and the accessible fraction of fluorophores for succinimide when compared to acrylamide, suggests that the tryptophan residues of TDSL are at least partially buried in the protein interior. The ionic quenchers, I and Cs+, being charged, can not diffuse, unlike the neutral quenchers, within the hydrophobic core of proteins and thus do not probe buried tryptophan residues. This makes them especially useful to study the environment of surface or exposed tryptophan residues [15,17]. For native TDSL, as can be seen from Tables 1 and 2, fluorescence quenching by these quenchers is substantially small and shows small quenching parameters. On the other hand, for denatured lectin, where the polypeptide chain is unfolded and tryptophan residues are exposed to the bulk solvent, the fluorescence quenching parameters obtained with these ionic quenchers increase significantly. This clearly gives further support to the conclusion drawn from an analysis of the emission spectra (see Section 3.1), that the tryptophan residues of native TDSL are buried in its hydrophobic core. Two more comments can be made here about the differences in quenching parameters between native and denatured lectin. Firstly, the increase in fluorescence quenching parameters of TDSL with Cs+, upon denaturation of the lectin, is not as large as those observed with other quenchers, especially with I (see Table 2). Based on this observation, one can infer the presence of at least one positively charged amino acid residue, under the experimental conditions, close to one or more tryptophan residues. Similar observations were reported for another seed lectin from Cucurbitaceae, TCSL [18]. Secondly, only for acrylamide, denaturation of TDSL results in complete accessibility of the quencher to the fluorophores (fa value equal to 1.0, linear dependence of F0/F on [Q], and nearly equal values of KSV and Ka). This most likely results from the neutral character and high efficiency of acrylamide as a protein fluorescence quencher. The inability of succinimide to achieve 100% accessibility suggests the presence of some local order in the protein polypeptide chain even in the presence of 6 M Gdn.Cl, which allows only partial accessibility to most likely one Trp residue. As mentioned in Section 3.1, this residual order in the protein structure is most likely due to the presence of disulfide bonds in the protein. Since acrylamide could achieve 100% accessibility, this local order in the protein structure does not prevent it from getting close to the Trp residue that is partially shielded from succinimide. Lactose binding to TDSL leads to a significant decrease of its fluorescence quenching parameters with all quenchers except acrylamide. This effect of sugar binding on the fluorescence quenching of TDSL
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suggests that the bound sugar makes it harder to the bulky quencher, succinimide as well as to the ionic quenchers (I and Cs+) to diffuse into the hydrophobic core of the lectin. The reason why quenching by acrylamide is not affected by bound sugar could be again the neutrality (compared to I and Cs+), size and efficiency (compared to succinimide) of this quencher. Sugar binding to two other Cucurbitaceae lectins, SGSL [17] and TCSL [18], did not significantly alter the fluorescence quenching by external quenchers. However, for another Cucurbitaceae seed lectin, Momordica charantia lectin, MCL, various quenchers showed decrease in fluorescence quenching parameters upon binding of a specific sugar, lactose [20]. These different effects of sugar binding by these similar lectins on their fluorescence quenching behavior can be explained in view of the amino acid residues involved in their sugar binding activity. In MCL, chemical modification studies show that both tryptophan and tyrosine are involved in the sugar binding activity [21]. Therefore, the presence of a specific sugar in the quenching medium is likely to provide some protection to the fluorescent Trp residues against the quenchers leading to smaller quenching parameters. The same can be said for TDSL, in which tyrosine, whose excitation energy usually is transferred to tryptophan, is involved in the lectin activity [4]. On the other hand, in both SGSL and TCSL, neither tryptophan nor tyrosine are involved in the sugar binding [17,22,23]. Therefore, in the absence of significant ligand-induced conformational changes, which can lead to differences in the accessibility of the Trp residues, for these two proteins the fluorescence quenching is not expected to be altered by the presence a specific sugar, which was experimentally observed [17,18]. Time-resolved fluorescence studies showed the presence of two components in the fluorescence decay profiles of TDSL. There are at least two reasons for multiplicity of tryptophan lifetime in proteins: (a) the presence of tryptophan in several different rotamers that are quenched to different extents by the functional groups (peptide backbone and amino acid side chains) in the microenvironment surrounding the indole ring [24] (for this reason, even single-tryptophan proteins exhibit multiexponential fluorescence decay), and (b) the heterogeneity of the environments of various tryptophan populations in multitryptophan proteins, such as TDSL in this study. In summary, the fluorescence quenching studies reported here demonstrate that the tryptophan residues of TDSL are in a heterogeneous hydrophobic environment. Lactose, occupying the sugar binding site, hinders the accessibility of all quenchers (except acrylamide) to the interior of the TDSL. Fluorescence quenching parameters obtained with Cs+ (compared to the other quenchers, especially I) suggests the presence of one
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or more positively charged amino acid close to some of the tryptophan residues.
5. Abbreviations TDSL SGSL TCSL MCL PBS
Trichosanthes dioica seed lectin snake gourd (Trichosanthes anguina) seed lectin Trichosanthes cucumerina seed lectin Momordica charantia lectin 10 mM sodium phosphate buffer, pH 7.4, containing 150 mM sodium chloride and 0.02% sodium azide Gdn.HCl guanidinium chloride (guanidine hydrochloride) bME b-mercaptoethanol MebGal methyl-b-D-galactopyranoside Trp tryptophan
Acknowledgements This work was supported by a research project from the Department of Science and Technology (India) to MJS. NAMS was supported by a Research Fellowship from the Sanaa´ University, Yemen. The IBH 5000 time-resolved fluorescence spectrometer was supported by the DST (India). We thank Professor A. Samanta for generous access to the timeresolved fluorescence spectrometer and Mr. Sandeep Banthia and Mr. Rana Karmakar for help with measurements on it.
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