Interaction of bis(1-anilino-8-naphthalenesulfonate) with yeast hexokinase: a steady-state fluorescence study

J o u r n a l of

Photochemistry Photobiolog 2 and

B:Biology

ELSEVIER

J. Photochem. Photobiol. B: Biol. 47 (1998) 190-196

Interaction of bis(1-anilino-8-naphthalenesulfonate) with yeast hexokinase: a steady-state fluorescence study Haripada Maity *, Sitapati R. Kasturi Department of Chemical Sciences, Tata Institute of Fundamental Re~'earch. Homi Bhabha Road, Mumbai 400 005, India

Received 3 August 1998; accepted 2 December 1998

Abstract Bis(1-analino-8-naphthalenesulfonate) (bis-ANS) is a useful probe for hydrophobic areas on protein molecules and it has been proposed that it has a general affinity for the nucleotide binding site(s). There appear to be two different classes of binding sites for bis-ANS on hexokinase and these can be tentatively assigned as primary and secondary binding sites. The rate of binding of bis-ANS at the primary binding site is fast, whereas binding at secondary site (s) is slow. The slow increase in the fluorescence intensity on binding with bis-ANS is not due to conformational change in the enzyme, which may lead to the increase in the quantum yield of the bound dye. Circular dichroism measurements indicate that there is no significant change in the secondary structure on binding with this probe. In the presence of saturating amounts of glucose, the increase in fluorescence intensity due to binding at the secondary binding site(s) is significantly lowered. This indicates that glucose-induced conformational change has been sensed by this probe. From kinetic studies, it has been observed that bis-ANS is an effective competitive inhibitor of yeast hexokinase with respect to ATP. The stoichiometry of binding of this fluorescent probe is about one per subunit at the primary site both in the presence and absence of glucose, and the dissociation constant of bis-ANS is unaffected by glucose. It is possible to decrease significantly the amount of fluorescence intensity at the primary site by nucleotides. These results indicate that bis-ANS interacts at the site where nucleotide interacts. Energy transfer experiments indicate the proximity of some tryptophan(s) and bound bis-ANS molecule(s). © 1998 Elsevier Science S.A. All rights reserved. Keywords: Hexokinase; Bis-( 1-anilino-8-naphthalenesulfonate);Fluorescence; Energytransfer; Quenching

1. I n t r o d u c t i o n

Mg + 2/Hexokinase

ATP + D-glucose Bis(1-anilino-8-naphthalenesulfonate) (bis-ANS) is a dimer of 1-anilino-8-naphthalenesulfonate. It is a better probe than 1,8-ANS for monitoring the hydrophobic areas on protein molecules and, in some cases, its binding affinity is orders of magnitude higher than that of 1,8-ANS [ 1-4]. Bis-ANS is almost non-fluorescent in aqueous medium but shows fluorescence when bound to proteins. It has been used to probe the structure-function relationship in several proteins, such as tubulin [5], myosin [4], lactic dehydrogenase [6], RNA polymerase [ 7 ], protein kinase [ 8 ], etc. Because of its shape, it has been proposed that bis-ANS might have a general affinity for ATP binding sites [4]. Yeast hexokinase (EC 2.7.1.1, ATP: D-hexose 6-phosphotransferase) catalyses the transfer of the phosphoryl group to the sixth hydroxyl group of glucose according to the reaction * Corresponding author. Present address: Department of Chemistry, Universityof Mississippi, University,MS 38677, USA.Fax: + 1-601-232-7300; E-mail: [email protected]

ADP +

D-glucose-6-phosphate Yeast hexokinase is structurally well characterized [ 9-13 ] and it is a homodimer with a molecular mass of 104 kDa [ 14 ]. The two subunits of the dimer have been found to be associated in a heterologous rather than an isologous fashion [ 1511, and these subunits are related by a 156 ° rotation and about a 13.8 A translation along the molecular axis [ 13 ]. The heterologous association of the dimer re,;ults in non-equivalent environments for the two subunits and consequently the two chemically equivalent subunits become non-equivalent with respect to the substrate binding. On the other hand, solution studies predict that the subunits of the dimer associate in a homologous fashion and these are equivalent with respect to the substrate binding [ 16]. The homologous association of the two identical subunits has been observed for most oligomeric proteins whose three-dimensional structures are known [ 17 ]. The tertiary structures of the monomer and the two subunits are similar [ 13]. However, each subunit

1011-1344/98/$ - see front matter © 1998 Elsevier Science S.A. All rights reserved. PIISlOll-1344(98)OO222-X

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H. Maity, S.R. Kasturi / J. Photochem. PhotobioL B: Biol. 47 (1998) 190--196

consists of two domains of roughly equal size and these are separated by a deep central cleft containing the sugar binding site; only one sugar binds per monomer and it binds to the same place for both the monomer and dimer [ 15,18 ]. When glucose binds, a hinge-bending movement occurs, which brings the two domains together and partially closes the cleft around the sugar molecule [ 13]. This substantial change in the conformation enhances the binding of the metal-ATP substrate and apparently protects the activated ATP from attack by water [ 19]. There is one nucleotide binding site per monomer of the enzyme molecule [ 20]. As bis-ANS has general affinity towards the ATP binding site [4], we have utilized this property to study the interaction of this dye with yeast hexokinase in the dimer form in detail using the steadystate fluorescence method. Here, we report our studies on the interaction of bis-ANS with yeast hexokinase. The objective has been to characterize the number of dye binding site(s) (n), the dissociation constant of the dye from the enzyme (Kd) and the response to the substrate-induced conformational change, if any, in the enzyme. Also, it is interesting to examine whether the dye binding affects the activity of the enzyme or brings about any conformational change in the enzyme. 2. M a t e r i a l s a n d m e t h o d s

2.1. Materials

Yeast hexokinase type C-302, ATP, ADP, NADP, glucose6-phosphate, glucose-6-phosphate dehydrogenase and Tris were obtained from Sigma, USA. Bis-ANS was obtained from Molecular Probes Inc., USA. All other chemicals were of analytical grade. 2.2. Sample preparation

Hexokinase obtained from Sigma was dialysed against 25 mM Tris-HC1, pH 6.4, until it was free from sulfate. The enzyme used in all the experiments reported here had a specific activity of 270-280 units m g - J. The concentration of hexokinase was determined by the measurement of absorption at 280 nm using the specific absorptivity of 1 ml m g - t cm ~ [21]. All the experiments described here have been done at 22°C and under conditions where hexokinase exists in the dimer state, unless mentioned otherwise. Hexokinase concentrations are defined as subunit concentrations based on a molecular mass of 52 000 Da. According to Lazarus et al. [22], the ratio of the maximum rate of phosphorylation of fructose to that of glucose ( F / G ) is very different for the two isoenzymes (PI and PII). The F / G ratio for the enzyme used in our experiments was found to be 1.3 and hence this enzyme has the characteristics of PII isoenzyme. 2.3. Enzyme assay

The activity of hexokinase was assayed spectrophotometrically by coupling with glucose-6-phosphate dehydrogenase

191

and the reduction of NADP was monitored at 340 nm [23]. The activity was measured at 25°C under the following conditions: 32 mM glucose, 2 mM ATP, 10 mM MgC12, 50 mM Tris-HC1 (pH 8.0), 0.4 mM NADP, and about 1 unit of glucose-6-phosphate dehydrogenase. One unit of enzyme activity is defined as the amount of enzyme that phosphorylates one micromole of glucose per minute under the above conditions. 2.4. Inhibition o f hexokinase

The procedure for the assay of hexokinase activity is described in Section 2.3. The hexokinase activity assay consists of two stages, namely, the catalysis of the phosphorylation of glucose by hexokinase and the reduction of NADP ÷ by glucose-6-phosphate dehydrogenase. The decrease in the enzyme activity by bis-ANS could be at either or both of these steps. In order to check this, the effect of bis-ANS in the reduction of NADP + by glucose-6-phosphate dehydrogenase was monitored and it has been found that bis-ANS also behaves as an inhibitor for the coupling enzyme, glucose6-phosphate dehydrogenase. For this reason, in order to monitor the effect of bis-ANS on the activity of hexokinase, sufficient glucose-6-phosphate dehydrogenase was added so that its inhibition was not a factor at the levels of hexokinase assay. Under this condition, the hexokinase activity, i.e., the phosphorylation of glucose, was still determining the rate and any inhibition by bis-ANS for hexokinase activity could be attributed to the effects on hexokinase itself. 2.5. Fluorescence measurements

The concentration of bis-ANS was monitored spectrophotometrically at 385 nm using a molar extinction coefficient of 16 790 M - ] cm-1 [24]. Since the binding of bis-ANS was found to be not instantaneous, bis-ANS and hexokinase were incubated for 2 h before the fluorescence measurements were made at equilibrium. At this point, it was observed that the binding of bis-ANS with hexokinase was almost complete. For the measurements of the fluorescence intensity of bound bis-ANS, samples were excited at 385 nm and the emission was measured at 500 nm in a cuvette of 0.3 cm path length. For energy transfer experiments, samples were excited at 295 nm. Experiments were performed with a slit width of 2.26 nm at both excitation and emission sites unless otherwise specified. All experiments were performed at 22°C. 2.6. Binding parameters

The stoichiometry and the dissociation constant for the binding of bis-ANS with hexokinase were determined according to the method developed by Klotz [ 2,3,25,26]. Let us consider a ligand (L) binding with an enzyme (E) to form a complex EL according to the reaction E+L=EL The dissociation constant (Kd) can be defined as

(1)

192

H. Mai~, S.R. Kasturi / J. Photochem. Photobiol. B: Biol. 47 (1998) 190-196

[E][L] Kd-- - [EL]

(2)

Assuming that there are n independent binding sites of bisANS per enzyme molecule and that the quantum yield of each site is the same, Eq. (2) can be written as K0 =

(n[E,]-[EL])[L]

(3)

[ELl

where (n [ E,) - [ EL ] ) denotes the number of sites available for binding and [EJ is the total concentration of the enzyme. Eq. (3) can be written as [Et]

[EL]

Kd 1 + niL] n --

(4)

is binding to the hydrophobic regions on the protein (Fig. 1 ) [ 27 ]. Binding of bis-ANS with hexokinase results in a timedependent increase in the fluorescence intensity (Fig. 2). On addition of bis-ANS to hexokinase there is an instantaneous increase in the fluorescence intensity and after that, a slow increase in the intensity was observed. The instantaneous increase in the intensity has been assigned to the binding at the high-affinity primary site (s) of hexokinase and similarly the slow increase in the intensity is attributed to the binding of bis-ANS at low-affinity secondary binding site (s). A similar time-dependent increase in fluorescence intensity in the interaction of bis-ANS with myosin [4] and tubulin [28] has also been reported. We have also observed a similar type of behaviour in the case of lysozyme (data not shown) in

Again, ILl = [Lt] - [EL] and here [LJ is the total concentration of the ligand. Substituting [L] in Eq. (4), we get [Et]

[EL]

Kd

+

35

1

(5)

-

n [ L , ] ( 1 - [-~j/[EL]/ n

30

~,

Again it is possible to show that Fobs [EL]= ~ [Lj

(6)

where Fobs is the fluorescence intensity at a given concentration of the ligand, and Fma., is the maximum fluorescence when all the ligand is bound to the enzyme. Substituting for [EL] from Eq. (6) in Eq. (5), we get [Et]

Kd

40

1

+ F~a----~[LJ n[L,l 1- Fob~ n

25

-8 2o 8ffl

10 5

J (7)

Eq. (7) was used for the determination of Ka and n. Fm,~ can be calculated from the plot of 1/Fob~ versus 1/[Et]. At higher concentration of bis-ANS, there will be a severe inner filter effect. So for the determination of binding parameters, the concentration of the dye was kept constant and the concentration of the enzyme was varied. Then a plot of

0

400

(b) i

i

i

i

i

i

425

450

475

500

525

550

Fig. 1. Fluorescence emission spectrum of 17.4 p~M bis-ANS in the presence of 9 ~M enzyme (a) and that of 17.4 IxM bis-ANS alone (b). The samples were in 25 mM riffs, pH 6.4.

24 23 22

[gt] F,,,,~-~[L,]

1

versus

[L,I 1 - Fob~

575

Wavelength (nrn)

E

_q

0

0

0

0

21

~o 20 e'-

was made. From the slope and intercept of the plot, the values of Ko and n were determined.

~

19

~

18

o E

17 16

3. Results and diseussion

3.1. Binding of bis-ANS to hexokinase Bis-ANS has very weak fluorescence emission with a ~tma x of about 515 nm and the emission maximum blue shifts to 490 nm upon binding to the protein, indicating that the dye

15

i

5

10

T

i

15

20

i

25

30

35

40

Time (min) Fig. 2. Time-dependent binding of bis-ANS with hexokinase in the absence ( O ) and presence ( • ) of glucose in 25 mM Tris, pH 6.4. The concentrations of bis-ANS, hexokinase and glucose in the solution were 17.4 IxM, 9 txM and 25 raM, respectively.

H. Maity, S.R. Kasturi / J. Photochem. Photobiol. B: Biol. 47 (1998) 190-196

addition to both the isoenzymes of hexokinase. Prasad et al. [28] suggested that the immediate increase in the fluorescence intensity observed in tubulin is due to the binding at primary binding site(s) and the equilibrium intensity (i.e., that obtained after about 2 h of incubation) is due to the binding at the secondary site(s). The slow increase in the intensity may arise from the slow conformational change in the enzyme on binding with bis-ANS, which may lead to the change in the quantum yield of the bound dye according to the reaction Slow

conforrnational

E+nL=(EL,~)

change

~

(EL,,')

But this possibility has been ruled out by an experiment where the concentration of bis-ANS was kept constant while the concentration of the enzyme was varied. At each concentration of the enzyme, the instantaneous and equilibrium fluorescence intensities were measured. The difference between these two intensities (i.e., instantaneous and equilibrium) was attributed to binding at the secondary binding site(s). The percentage change in the intensity due to binding of bisANS at the secondary binding site(s) was found to decrease with increasing concentrations of the enzyme (Fig. 3). This experiment has been performed both in the absence and the presence of glucose, and similar types of behaviour were observed (Fig. 3). If the slow increase in the intensity is due to the conformational change on the enzyme on binding of bis-ANS at the primary site, the percentage change in the secondary fluorescence should have remained constant. This indicates that the slow increase in the intensity is not due to the conformational change but to the interaction of bis-ANS at different site (s) (assigned as secondary binding site(s)) and the rate of binding at this site(s) is slow. In the presence of glucose, i.e., in the 'closed' conformation of the enzyme, the increase in the secondary fluorescence intensity was sig40

220 0

200

35

c~ 180 ~

160

•~

140

0

3o

0

25

--~ 120 t,L

20

.c_ 100 rc~ -

80

~L) o~

60

0

0

o

2o 0

nificantly less for a given period of time (Fig. 2). This observed decrease in the presence of glucose may either be due to a decrease in the available secondary binding site(s) on the enzyme or to a decrease in the quantum yield of the bound bis-ANS at the secondary binding site(s), or else the rate of binding of bis-ANS at the secondary binding site(s) may be slower in the presence of glucose. Such an observed change indicates that the binding of the dye is sensitive to the conformational changes induced by the binding of glucose to the enzyme. The effect of binding of bis-ANS and glucose on the circular dichroism (CD) spectrum ofhexokinase has been studied. On addition of bis-ANS, the CD spectrum of the enzyme in the far-UV region was unperturbed. There was no timedependent alteration of the CD spectrum. Binding of glucose also did not alter the CD spectrum. This indicates that binding of glucose and bis-ANS does not lead to any significant change in the secondary structure of the enzyme. These observations alone cannot rule out the possibility of any conformational perturbation in the random coil regions and it has been proposed that glucose-induced conformational change is localized in the random coil region [29].

3.2. Binding parameters for the interaction of bis-ANS Fig. 4(a) shows the binding isotherm of the interaction of bis-ANS with hexokinase at the primary binding site(s) in the presence and absence of glucose. In this titration, the concentration of bis-ANS was kept constant and the concentration of hexokinase was varied. In the reverse titration, where the bis-ANS concentration is varied, there will be a severe inner filter effect at higher dye concentration and hence the reverse titration was not carried out. An attempt has been made to obtain the binding parameters (n and Kd) at the primary binding site(s) with the assumption that the binding site(s) are independent. The binding parameters n and Kd were determined according to Eq. (7) as described in Section 2.6. Analysis of the binding isotherm in the absence of glucose (Fig. 4(a) ) according to Eq. (7) gives Kd = 23 _ 3 I~M and n = 1.2 per subunit (Fig. 4(b) ). In the presence of glucose, a Kd value of about 26 ~M and n = 1 for the binding of bis-ANS at the primary binding site of hexokinase were obtained. The value of Kd obtained here is very close to that of Ki obtained in the inhibition studies (see Section 3.4).

0

4O

0

193

F

I

I

I

10

20

30

40

50

3.3. Effect of the nucleotide and glucose on the fluorescence of bound bis-ANS

8

o I

I

60

70

80

[Hexokinase] ~¢1 Fig. 3. Variation of fluorescence intensity due to the binding of bis-ANS at the secondary binding site(s) as a function of hexokinase concentration in 25 mM Tris, pH 6.4, both in the presence (O) and absence ((3) of glucose. The concentrations ofbis-ANS and glucose were 16 txM and 15 mM, respectively, and the concentration of the enzyme was varied from 2.8 to 72 p.M. Other details are given in the text.

In order to ascertain whether the dye binds to the nucleotide binding site on hexokinase, we have studied the effect of ADP on the fluorescence intensity of the bound bis-ANS molecule(s) qualitatively. The fluorescence intensity was monitored at each step of the addition of the nucleotide. In a titration, ADP concentration was varied and with increasing concentration of ADP, the fluorescence intensity decreases (Fig. 5). The effect of ADP on the fluorescence of bound

H. Mai~, S.R. Kasturi/J. Photochem. PhotobioL B: Biol. 47 (1998) 190-196

194

,81

32

(a)

16

30 0

140

12 i ¢11 10 kl.0

o

28

•

0 0

26 0 0 ii

8

Oe

6

0

o~

24

0

0 0 0

22

o

0

0

18

2

0

0

"r

I

I

I

I

0

20

40

60

80

16 1O0

0

I

I

I

I

5

10

15

20

Fig. 5. Effect of ADP on the fluorescenceintensity of hexokinase-bis-ANS complex in 25 mM Tris, pH 6.4. Hexokinase was incubated with bis-ANS for 2 h before this experiment was performed. The concentrations of bisANS and hexokinase were 17.4 and 9 p,M, respectively. The concentration of AI)P was varied from 0.7 to 21.6 raM.

3.5

(b)

3.0 ~

E

%2.s 2.0

o~

1.5

Z

o

o.5 0.0

25

[ADP] (raM)

[Hexokinase] pM

%

0

20

4

u.°

0

r

i

i

i

i

0.00 0.04 0.08 0.12 0.16 0.20 0.24 l/[bis-ANS]o(1 - Fobs/Fmax)

Fig. 4. (a) Binding of bis-ANS to hexokinase dimer at the primary site(s) as a function of enzyme concentrationin the absence (©) and in the presence (O) of 15 mM glucose. The sample contained 16 I~M bis-ANS in 25 mM Tris, pH 6.4. The enzyme concentration was varied from 2.8 to 72 IxM.The intensity was measured immediatelyafter the addition of bis-ANS.For each measurement, separate samples were prepared. (b) Plot of [E,]/(F~,bJ F,,1.~)[L~] vs. 1/ [LJ ( 1- F,,bJF,,.,Ofor the complex hexokinase-bis-ANS for the determination of Kd and n. [ bis-ANS] o is the initial concentrationof bis-ANS. bis-ANS has been studied both in the absence (Fig. 5) and presence (not shown) of glucose and the results obtained were approximately similar. The decrease in the fluorescence intensity can be attributed to the displacement of the bound dye by the nucleotide. It is observed that the decrease in fluorescence intensity on the addition of nucleotides was instantaneous. But a slow increase in the intensity was observed with time. As the binding of bis-ANS at the primary site was fast, it is expected that the displacement o f b i s - A N S by the nucleotide will be fast. This instantaneous decrease in fluorescence also supports the idea that nucleotides are interacting at the primary site. The slow increase in intensity with time in the presence of A D P is probably due to the fact that the free dye concentration increases on the displacement of bis-ANS. Then the displaced dye further interacts at the secondary site, which results in a slow increase in the fluorescence intensity. At a saturating concentration of glucose ( 10.4 m M ) the total fluorescence intensity was reduced only by about 13%.

Though this small decrease in fluorescence intensity was fast, a further slow decrease in fluorescence intensity was also observed. The fast decrease in the fluorescence intensity is probably due to the change in the conformation of the enzyme on binding with glucose, because from the inhibition studies we have not observed any significant inhibition by bis-ANS with respect to glucose. But inhibition has been observed when the enzyme was incubated with bis-ANS for 15-20 rain. It may be possible that bis-ANS also binds to the glucose binding site where the binding is slow, and consequently the dissociation will also be slow.

3.4. Effect of bis-ANS on hexokinase activiD' Displacement of bis-ANS from the enzyme by A D P indicates that the primary binding site o f bis-ANS is most likely the active site. Such a model would predict that in a kinetic experiment, bis-ANS must act as a competitive inhibitor with respect to ATP. Addition of bis-ANS to hexokinase results in the inhibition of enzyme activity. Bis-ANS also inhibits the glucose-6phosphate dehydrogenase activity used as a coupling enzyme in the hexokinase assays. Care was taken to make sure that in coupled assays, dehydrogenase activity was not the ratelimiting one. Hexokinase activity was measured at two fixed concentrations of bis-ANS with varying A T P concentrations (0.06-0.44 raM). The data are plotted as a Lineweaver-Burk plot (Fig. 6). For a competitive inhibition, 1

= K.pp

Vo

1

1 + -Vm,x [ s ] vmo~

(8)

where [ [I1 K~pp=Kmtl+ -~ )

(9)

195

H. M a l t y . S.R. K a s t u r i / J . P h o t o c h e m . P h o t o b i o l . B : Biol. 4 7 ( 1 9 9 8 ) 1 9 0 - 1 9 6

60

30

54

25

48 20

,e-

•~ 42 c

15

~

10

-8 30

36

"~

~ 24

5

e

0

,."

",

0

0

I

I

I

[

2

4

6

8 10 12 14 16 18

I

~

I

[

•"/d' "'"~ ..

12

I/[ATP] mM"1

Fig. 6. Lineweaver-Burkplot. The activity of the enzyme was measured at different concentrations of bis-ANS, 0 txM (O), 14.3 I~M (©) and 19 ~M (A). The concentrationof ATP was varied between 0.061 to 0.435 mM. Other details are given in the text. where Vo is the initial velocity, Vmaxis the maximum velocity, IS] is the substrate concentration, [I] is the inhibitor concentration, Km is the Michaelis-Menten constant and K~ is the inhibition constant. Intersection of all the lines at a single point on the y axis implies competitive inhibition of the enzyme activity by bis-ANS with respect to ATP. The value of Km obtained for ATP in the absence of bis-ANS was 0.13 mM, which is in good agreement with that reported in the literature [30]. At 14.3 and 19 p,M of bis-ANS, the K, pp values for ATP were 0.18 and 0.23 mM, respectively. Using these values in Eq. (9) along with the value of K,, for ATP in the absence of inhibitor, the Kj = 28 + 5 ~M was obtained. Hence bis-ANS is an effective competitive inhibitor with respect to ATP. Bis-ANS was found to be a very poor inhibitor of hexokinase activity with respect to glucose. In the presence of 3 mM ATP, 47 txM glucose and 20 txM bis-ANS, only about 4-5% inhibition was observed. This suggests that instantaneous binding of bis-ANS does not involve the glucose binding site. But when the enzyme was incubated for about 15-20 min, the reaction was inhibited very effectively. Analysis by using the Michaelis-Menten equation requires that the reaction between inhibitor and free enzyme or enzyme-substrate complex should be fast and reversible [ 31 ]. Inhibition studies were done for the binding of bisANS with hexokinase at the primary site where the binding was fast.

3.5. Energy transfer from enzyme tO'ptophan(s) to bound bis-ANS There are four tryptophan residues per subunit and these are located in a non-polar environment as the emission maximum occurs at 328 nm when the enzyme was excited at 295 rim. The emission spectrum of hexokinase (4 .... = 328 nm) overlaps with the absorption bands of bis-ANS. This overlap indicates that the energy transfer from tryptophan residues to bound bis-ANS is possible. For the energy transfer study,

I

I

I

J

I

350

400

450

500

550

Wavelength (nm) Fig. 7. Concentration dependence of bis-ANS in the energy transfer between tryptophan(s) of hexokinase and bound bis-ANS molecule(s) in 25 mM Tris, pH 6,4. For a fixed concentration of hexokinase (9 i.zM) the concentration of bis-ANS was varied: (a) 0 IxM; (b) 3.9 txM (c) 7.8 ~M (d) 11.6 o,M and (e) 15.4 IxM. Excitation was done at 295 nm and the emission spectra were recorded from 305 to 575 nm.

excitation was done at 295 nm. Bis-ANS has an absorption band at 328 nm which is the emission maximum of tryptophan. When bis-ANS was added to hexokinase and excited at 295 nm, a decrease in emission at 328 nm and the appearance of a maximum at around 500 nm, characteristic of bound bis-ANS, was observed. Fig. 7 shows this result in which a fixed concentration of the enzyme was titrated with increasing concentrations of bis-ANS. These experiments were performed in the samples where the enzyme was incubated with bis-ANS for 2 h. We have also monitored the time-dependent increase in the energy transfer (not shown). These results indicate that at least a few of the tryptophan residues are located within the energy transfer distance from bound bisANS. Distance measurement in such cases is not possible due to the presence of multiple tryptophan residues and the presence of bis-ANS at tire primary and secondary binding sites. But it does indicate the proximity of some tryptophan(s) to the dye binding site.

4. A b b r e v i a t i o n s

Bis-ANS Tris

bis(1-anilino-8-naphthalenesulfonate) tris(hydroxymethyl)-aminomethane

Acknowledgements

We thank Professor Gotam K. Jarori for his critical comments and useful discussions on the manuscript. We also thank Professor B.S. Prabhananda for many useful discussions.

196

H. Malty, S.R. Kasturi / J. Photochem. Photobiol. B: Biol. 47(1998) 190-196

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[ 16]

[17] [ 18 ]

[ 191

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