Fluorescence study of fungal lipase from Humicola lanuginosa

Journal

of

Photochemistry and Photobiology B:Biology

ELSEVIER

Journal of Photochemistry and Photobioiogy B: Biology 45 (1998) 95-102

Fluorescence study of fungal lipase from Humicola lanuginosa A. Stobiecka a,,, S. Wysocki a, A.M. Brzozowski b aInstitute of General Food Chemistry, Technical University, Stefanowskiego 4/10, 90-924 t_~d~, Poland b Department of Chemistry, University of York, York, Y01 5DD, UK Received 12 November 1997; accepted 30 June 1998

Abstract

Time-resolved and steady-state fluorescence quenching measurements have been performed to study two different conformations of the fungal lipase from Humicola lanuginosa. The intrinsic fluorescence of tryptophan Trp89 residue, located in the 'lid' region, has been used as a probe for the dynamics of protein. The native ( 'closed-lid' ) form of the enzyme has been found to decay as a triple exponential with time constants and relative contributions of 5.4 ns (74.3%), 2.2 ns (20.4%) and 0.4 ns (5.3%). A comparison of recovered decay parameters obtained for native and mutated H. lanuginosa lipase shows that Trp89 contributes about 61% to the class of emitting species with the lifetime of 5.4 ns. The fluorescence quenching data show that three out of four tryptophans (i.e., 117, 221 and 260 residues) within H. lanuginosa lipase are totally quenchable by acrylamide while completely inaccessible to iodide. On the contrary, the Trp89 residue is available for both quenchers. Using steady-state iodide fluorescence quenching data and the fluorescence-quenching-resolved-spectra (FQRS) method, the total emission spectrum of the native lipase has been decomposed into two spectral components. One of them, unquenchable by iodide, has a maximum of fluorescence emission at 330 nm and the second one, exposed to the solvent, emits at 338 nm. The resolved spectrum of the redder component corresponds to the Trp89 residue, which participates in about 65% of the total H. lanuginosa emission. The dynamic SternVolmer quenching constants calculated for both native ( 'closed-lid' ) and inhibited ('open-lid') lipase are 2.71 and 4.49 M - ~, respectively. The values obtained indicate that Trp89 is not deeply buried in the protein matrix. Our results suggest that distinct configurations of fungal lipase can be monitored using the fluorescence of the Trp89 residue located in the 'lid'-helix which participates in an interfacial activation of the enzyme. © 1998 Elsevier Science S.A. All rights reserved. Keywords: Humicola lanuginosa lipase; Conformation; Fluorescence

1. Introduction

The fluorescence spectroscopy of tryptophan residues in peptides and proteins has provided considerable information on biomolecule structure and dynamics. The fluorescence lifetime and quantum yield of tryptophan in protein depend on the physical properties of its local environment. It is assumed that fluorescence emission heterogeneity observed in multi-tryptophan-containing proteins has its origin in the different solvent exposure of a particular chromophore or in its specific interactions with a protein matrix [ 1-5 ]. The kinetic behaviour of lipases (triacylglycerol ester hydrolases EC 3.1.1.3) conducting biocatalysis in biphasic systems or in pure organic solvents has been extensively studied in recent years [6,7]. Lipases are highly versatile enzymes which are able to carry on a great variety of reactions [ 8-10 ]. The unique properties of lipases are connected with * Corresponding author. Tel.: + 4 8 (042) 631 34 26; E-mail: astobiec@ snack,lodz.pl

their molecular architecture. The fundamental aspect oflipase action concerns an interfacial activation phenomenon [ 11 ]. The stereochemistry of lipase activation was elucidated by crystallographic studies. According to the X-ray analysis of human pancreatic lipase (HPL) [ 12,13] and fungal lipases from Rhizomucor miehei [14-16], Geotrichum candidum [ 17 ], Candida rugosa [ 18,19 ] and Candida antarctica [ 20], the enzymatic mechanism of interracial activation involves conformational changes in the enzyme. These studies demonstrated that the activation of lipase was achieved by rearrangement of one or more helical loops which exposed the catalytic site to the solvent. The H. lanuginosa lipase belongs to a family of extracellular fungal lipases which preferentially hydro!yse fatty acids from the snl and sn3 positions of a triglyceride molecule. Derewenda et at. [21] solved the structure of the lipase at 1.8 A resolution. The three-dimensional structures ofH. lanuginosa-inhibitor complexes were refined by Brzozowski [22] and Lawson et al. [23]. The fungal acylhydrolase is a single-domain protein (mol. wt. = 30 000 Da) built on eight

1011-1344/98/$ - see front matter © 1998 Elsevier Science S.A. All rights reserved. PIlS 1 0 1 1 - 1 3 4 4 ( 9 8 ) 0 0 1 6 7 - 5

A. Stobiecka et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 95-102

96

central, twisted parallel, [3-sheet backbones connected by orhelices. The active site is composed of a Ser146-His258Asp201 triad. The lipase contains four tryptophans, 10 tyrosines and 15 phenylalanines per molecule. Structural Trp 117, Trp221 and Trp260 are not involved in the interfacial activation process. Trp89 is located in the short a-helix, corresponding to the 'lid' that covers the active centre. It has been demonstrated experimentally that Trp89 is essential for enzyme activity [24,25]. The results of a crystallographic analysis of H. lanuginosa diethylphosphate and H. lanuginosa--dodecylethylphosphonate complexes together with the molecular dynamics simulations [ 23 ] indicated a high mobility of Trp89 side chain during the interfacial activation. In this paper we report for the first time the results of a fluorescence study obtained for H. lanuginosa lipase. However, as the fluorescence decay of lipase that contains four tryptophan residues can be analysed in terms of three classes of emitting species, we demonstrated that the emission from Trp89 residue might be resolved using fluorescence quenching measurements.

2. Experimental The highly purified lyophilized samples of native, inhibited and mutated lipase from H. lanuginosa were a generous gift from Novo Nordisk A/S (2880 Bagsvaerd, Denmark). We used a three-tryptophan-containing mutant of the enzyme in which Trp89 was substituted by phenylalanine. The inhibited lipase was obtained by complexing the enzyme protein with phenylmethylsulfonyl fluoride (PMSF, a typical serine protease inhibitor). The best commercially available research-grade chemicals were used and were supplied by Sigma (potassium iodide, sodium chloride, sodium thiosulfate, Tris-HC1) and Merck (acrylamide). All measurements were performed in 0.05 M Tris-HCl buffer, pH 7.20, containing 0.1 M NaC1, prepared in twicedistilled water. The protein solutions had an initial absorbance lower than 0.1 at the excitation wavelength. Fluorescence lifetime measurements were made at 25°C using a FL900CDT single-photon pulse fluorimeter from Edinburgh Analytical Instruments. For all experiments the excitation wavelength was 295 nm and the excitation and emission ban@asses were 3.6 nm. The instrument response function was recorded by collecting scattered light from a Ludox silica suspension. Fluorescence decay from both a sample and scattering solution was acquired to 1.0× 104 counts in the peak. The counting rate was less than 2% of the lamp repetition rate. Fluorescence decays were fitted to a sum of exponentials:

t

~,[R(t)-R(t-At)] 2 DW= ~'

, ~[R(t)] 2

(2)

0

where R(t) are the weighted residuals. The Durbin-Watson parameter is a measure of the correlation between the residual values in neighbouring channels. A maximum in this parameter implies maximum randomness of residuals. Steady-state fluorescence measurements were performed with a Perkin Elmer LS50B spectrofluorimeter. The sample holder was thermojacked and all experiments were done at 21 + 0.5°C. At excitation wavelengths ranging from 290 to 305 nm the excitation bandwidth was set to 2.5 nm. The emission bandwidth ranged from 2.5 to 4 nm. Fluorescence quenching experiments were performed by adding microlitre amounts of acrylamide or iodide concentrated solution directly to the protein sample in the cuvette. KI solutions were freshly prepared before each measurement and contained a trace of sodium thiosuffate to retard I3formation. The correction for absorptive screening by acrylamide was performed using ¢295= 0.25 M - 1 c m - 1 The fluorescence quenching data were fitted to the modified version of the Stern-Volmer equation: F 0

~ f = i~= l ( l + K i [ Q ] ) e x p Vi[Q]

(3)

where Fo and F are the fluorescence intensities in the absence and presence of the quencher concentration [Q], Ki and Vi are dynamic and static quenching constants, respectively, and f~ is the fractional contribution of fluorescent component i. In order to resolve the fluorescence spectra of lipase into components, we used the fluorescence-quenching-resolvedspectra (FQRS) procedure of Stryjewski and Wasylewski [27]. Each analysis was performed for about 40 potassium iodide quenching data sets obtained over the wavelength region 310-400 nm for excitation at 295 nm. The parameters of Eq. (3) were calculated with the iterative non-linear leastsquares method.

3. Results and discussion

n

l(t) = ~ otiexp(--t/'ri)

The parameters describing the true sample decay function were extracted from the experimental data by a non-linear least-squares convolution process. The goodness of fit of the curves was judged by the reduced chi-squared (X2) criterion and by inspection of the systematic deviations in the weighted residuals and autocorrelation function, respectively. As an additional criterion of quality of fits the Durbin-Watson parameter [ 26] was used, which was calculated according to Eq. (2):

( 1)

i=l

with amplitudes ~i and decay lifetimes zi.

The fluorescence of tryptophan residues in the lipase from H. lanuginosa (HLL) and their quenching by acrylamide and

97

A. Stobiecka et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 95-102

1.0-

/

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330

300

~ 0.5,

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325 350 375 Emission wavelength [ nm ]

400

Fig. 1. Steady-state emission spectra of (- - -) HLL, (. - • ) oHLL lipase and ( ) W89F mutant in 0.05 M Tris-HC1buffer, pH 7.20 containing0.1 M NaC1at 2I°C. Excitation was at 295 nm. Inset shows the dependence of fluorescenceemission maximumon excitation wavelengthfor HLL (O), oHLL (&) and W89F lipase ( • ).

iodide were studied. The data were compared with those for inhibited ( o H L L ) and mutated HLL lipase (W89F). Despite differences in the primary structure and a number of tryptophan residues within the protein, the value of the fluorescence maximum of H. lanuginosa lipase is similar to those reported for HPL [28] and rice bran lipase [29]. The fluorescence peak of HLL and oHLL lipase in the buffered aqueous solution of pH 7.20 containing 0.1 M NaC1 when illuminated at 295 nm lies at 340 nm. W89F mutant emits fluorescence at 334 nm at the same conditions (Fig. 1 ). This observation indicates that the lack of Trp89 in W89F lipase has an important effect on the overall spectral distribution. In addition, when the excitation wavelength was changed from 290 to 305 nm, the excitation-dependent spectral shift was observed only in the case of W89F protein (Fig. 1 inset). Results obtained suggest that three (i.e., Trp117, Trp221 and Trp260) out of four tryptophans within the HLL protein are located in a more hydrophobic region of molecule in cornparison with Trp89 residue. The red shift of the emission spectra when excited in the long-wavelength absorption band is probably associated with dipole relaxation processes. A similar phenomenon was observed in single- and multitryptophan-con taining proteins [ 1,30]. Typical fluorescence decay curves measured at 25°C with hex = 295 nm and at the maximum of emission for the HLL and W89F lipases are shown in Fig. 2. The same experiments were also done with inhibited HLL lipase (Fig. 3). All data were analysed in terms of a multi-exponential decay law (Eq. (1) ). The quality of fit was judged by the magnitude of the reduced X2 criterion and the random distribution of both the weighted residuals and autocorrelation function obtained after deconvolution. The fluorescence lifetimes and fractional contributions of each component for lipases are presented in Table 1. The

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Time [ ns ]

Fig. 2. Fluorescence decays of HLL lipase (HI.J.,) and its mutant (W89F) excited at 295 nm and monitoredat 340 and 334 tun, respectively, at 25°C. The left curve is the reference decay. The solid lines are the best fit of the experimental data to a triple-exponentialdecay law (Eq. ( l ) ). See Table 1 for lifetimes. The weighted residuals plots are shown at the bottom of the curves. Inset: autoeorrelation function of the residuals calculated for fluorescence decay of HLL lipase.

98

A. Stobiecka et al. / Journal of Photochemistry and Photobiology B." Biology 45 (1998) 95-102

Table 1 Fluorescence decay parameters for W89F, HLL and oHLL lipases. The excitation wavelength was 295 nm and emission was 334 nm for W89F lipase and 340 nm for native and inhibited H. lanuginosa lipase, respectively. The values in parentheses are the standard deviations of lifetime

100000

O2[o0~L J , ~

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10000

Lipase "r] (ns)

f~ "

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(ns)

(%)

(ns)

(%)

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t/J r. ®

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W89F

2.17 71.30 (0.03)

HLL

5.37 74.31 2.16 20.43 0.40 (0.03) (0.10) (0.07)

5.26 1.092 1.75

oHLL

5.56 62.52 2.46 30.56 0.70 (0.04) (0.11) (0.09)

6.91 1.026 1.84

lO.

5.36 12.88 0.60 15.82 1.064 1.81 (0.14) (0.04)

"f,. is the relative contribution of component i calculated from the relationship:fi = (cti'6/Eictizi)100%. b D W is the Durbin-Watson parameter.

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the long-lifetime c o m p o n e n t of r i L L decay, r~ = 5.37 ns, contributes about 74.31%, while the shorter lifetime c o m p o n e n t rl = 2 . 1 6 ns contributes only 20.43%. A similar pattern of fluorescence lifetime distribution was observed for native and inhibited H L L lipase. In the latter case the contribution of the d o m i n a n t component, rl = 5.56 ns, was lower by about 12% in comparison with native lipase. This effect is probably caused by interactions b e t w e e n the Trp89 residue and P M S F inhibitor. The presented decay calculations for H L L and W 8 9 F lipase demonstrate that the presence of Trp89 residue dramatically increases the contribution of the long-lifetime c o m p o n e n t to the total emission. A comparison of the recovered decay parameters obtained for native and mutated lipase showed that approximately 61% of fluorescence in the native protein comes from Trp89. Although the information available with the lifetimes of individual tryptophan residues is still limited,

do

Time [ ns ] Fig. 3. Fluorescence decay of inhibited HLL lipase (oHLL) excited at 295 nm and monitored at 340 nm, 25°C. The left curve is the reference decay. The solid line is the best fit of the experimental data to a triple-exponential function (see Table 1 for lifetimes). A weighted residuals plot (bottom) and the autocorrelation function of the residuals (inset) are also shown. fluorescence decay of tryptophan residues in HLL, o H L L and W 8 9 F lipases was best described by a triple-exponential function with nearly the same three time constants but different fluorescence fractional contributions. The decay of the H L L m u t a n t is d o m i n a t e d b y the a'l = 2.17 ns component, which contributes about 71.30% of the emission. Contrary to W89F,

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Acrylamide [ M ] Fig. 4. Stern-Volmer plots for acrylamide-quenching of H. lanuginosa lipase and W89F mutant when excited at 295 nm, 21°C. (A) I-ILLprotein with emission at 340 nm and (B) W89F protein with emission at 334 nm. The solid lines represent least-squares fits with the parameters presented in Table 3 and Table 2 (model 5), respectively. The right panels show residuals plots of the fits.

A. Stobiecka et al. /Journal of Photochemistry and Photobiology B: Biology 45 (1998) 95-102

it is clear that Trp89 is the largest contributor to enzyme fluorescence emission. In order to obtain more information concerning the kinetic parameters of tryptophan residues in HLL, oHLL and W89F lipases, quenching studies were performed. The fluorescence quenching data were analysed according to the modified Stern-Volmer equation (Eq. (3)). During the analysis of each quenching data set, the values of Ki, Vi and f~ were allowed to run free. The Stern-Volmer plots of quenching of HLL lipase and its mutant by acrylamide are shown in Fig. 4. An upward curvature was the common pattern for each protein studied, which indicates a significant contribution of static quenching to the total fluorescence quenching performed with acrylamide (data obtained for oHLL lipase are not shown). The theoretical fits (Eq. (3)) to the experimental points were obtained as a result of subsequent analysis performed for different fluorescence quenching models. The best kinetic model was chosen using the following statistical criteria: the minimum of reduced X~ function, random residual distribution and the lowest standard deviation of each floating parameter (i.e., Ki, Vi and f/). Parameters describing the acrylamide-quenching of W89F protein are given in Table 2. When the data were analysed in terms of only one class of quenchable component without or with static quenching (models 1 and 2) and two classes of accessible components without or with static quenching (models 3 and 4, respectively), the reduced )(2 values were acceptable but higher in comparison to the three-component model. We decided to reject model 4, in which the standard deviation of the V2 value was higher than the calculated parameters themselves. The introduction of a fourth quenchable class of tryptophans did not improve the accuracy of the fit. The values of X2 and residuals distribution were very similar in the case of models 5 and 6, but the former was characterized by a lower standard deviation of the parameters obtained. Considering the criteria mentioned above, the quenching data of W89F lipase appeared to be well described by the threecomponent model with all tryptophans accessible to acrylamide. Although two small fractions in HLL mutant possess significant dynamic quenching constants (f2 = 0.08, Ksv2 = 9.80 M - ~ and f3 = 0.04, Ksv3 = 3.96 M - ~), the third main fraction is quenchable via both dynamic and static mechanisms (ft=0.88, K s w = 0 . 4 4 M - l and V~=0.34 M-l).

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The same fitting procedure was repeated when acrylamidequenching data for native and inhibited HLL lipase were analysed. The quenching parameters obtained for HLL and oHLL proteins together with the reduced X~ value and standard deviation values are presented in Table 3. In both cases three quenchable fractions were found. For HLL iipase two components were characterized by the static and dynamic constants (,#1=0.60, Ksv~ =0.78 M -~, V~=0.41 M - ; and f3 = 0.10, Ksv3 = 1.09 M - ~, V1 = 0.54 M - 1), while for inhibited HLL lipase only one class of tryptophans might be described with the static constant (fl=0.81, Ksv~=0.59

_.--,

X

I

100

A. Stobiecka et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 95-102

Table 3 Fluorescence quenching parameters for native and inhibited H. lanuginosa lipase. The excitation wavelength was 295 ran and emission was observed at 340 nm. The values in parentheses are the standard deviations of parameters obtained Lipase

f~

Acrylamide quenching HLL 0.60 (5.71×10 -2) oHLL 0.81 (1.97×10 -~)

K, (M -l)

V~ (M -u)

f2

K2 (M-')

f3

K3 (M -I)

V3 (M -I)

)(2

0.78 (1.88×10 2) 0,59 (9.75×10 -3 )

0.41 (8.18× 10 -3 ) 0.44 (7.64× 10 -3)

0.30 (3.00× 10 -2 ) 0.03 (1.71×10 -3)

1.13 (2.43× 10 -2 ) 20.80 (3.36×10 -2)

0.10 (2.22×10 -2 ) 0.16 (1.47×10 -2)

1.09 (5.79× 10 -2 ) 1.98 (1.07×10 -~)

0.54 (1.30×10 -J)

0.3377

0.65 (4.19× 10 -5) 0.48 (1.99× 10 -5)

2.71 (3.08× 10 -4) 4.49 (4.08 × 10 -4)

Iodide quenching HLL 0.35

0

oHLL

0

0.52

0.2624

0.4911 0.3910

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KI[M] Fig. 5. Stern-Volmer plots for iodide-quenching of native ( A ), inhibited (B) and mutated (C) 1-1.lanuginosa lipase when excited at 295 nm, 21 °C. The best fits of the quenching data obtained for HLL (A) and oHLL (B) protein are presented as solid lines with the parameters given in Table 3. The right panels show residuals plots of the fits.

M - l, V~= 0.44 M - 1). A comparison of acrylamide-quenching parameters obtained for mutated, native and inhibited HLL lipase indicates the presence of a common fraction which is quenchable by a collisional as well as a static mechanism. The values of the dynamic Stern-Volmer constants cited above suggest that the main component is quenched to a moderate degree, contrary to the second small component found in oHLL and W89F molecules with Ksv = 20.80 M and Ksv = 9.80 M - ~, respectively. Although there are a few references in the literature that concern the fluorescence of lipases, it should be stressed that the availability of fluorimetric measurements to study conformational changes in HPL was demonstrated by Ltithi-Peng and Winkler [28]. According to the results presented herein, an increasing accessibility of a single Trp252 residue, a side chain of the flexible flap, to the acrylamide quencher was induced upon the formation of an inhibitor-lipase complex. In contrast to the case of native and inhibited forms of H. lanuginosa lipase,

the acrylamide-quenching profile obtained for free and acylated form of HPL showed no significant deviations from linearity. Values of the dynamic constant and fractional contribution calculated for native and inhibited HPL were K = 3 M - l , f = 0.89 and K = 3.92 M - i , f = 0.95, respectively. As it was suggested, only one tryptophan residue, i.e., Trp 252, was quenchable by acrylamide, while the remaining six out of seven tryptophans within the HPL protein were not available for the quencher [28]. The Stern-Volmer plots of iodide-quenching of native, inhibited and mutated HLL lipase are shown in Fig. 5. In the case of HLL and oHLL enzyme, the observed downward curvature implies the presence of fluorescence components which have different accessibility to the quencher. On the contrary, the experimental data obtained for W89F lipase indicate that tryptophan residues within the mutant protein are not available for iodide. For native and inhibited HLL lipase the best fits (solid lines in Fig. 5) were obtained with

101

A. Stobiecka et aL / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 95-102

the two-component model in which one class of tryptophans was quenchable by KI, while the second one was totally inaccessible to the quencher. The results presented for iodide-quenching performed for the proteins studied can prove that the only single tryptophan residue, i.e., Trp89, is located near the protein surface. It may be assumed that the presence of lipase inhibitor covalently bound to the catalytic centre causes the displacement of short, flexible 'lid'-helix. Thus, it seems likely that Trp89 in the inhibited, 'open-lid' form of the enzyme becomes more exposed to the solute in comparison with the native, 'closedlid' form of HLL lipase. This hypothesis is in reasonable agreement with the calculated Stern-Volmer constants which have been obtained for iodide-quenching experiments. In the case of oHLL protein the component accessible to the KI was characterized by Ksv = 4.49 M - l, while in the case of native HLL lipase the value of the dynamic constant was about one and half times lower (Ksv = 2.71 M - J). The FQRS together with the dynamic quenching SternVolmer constants calculated for different emission wavelengths for native and inhibited HLL lipase are presented in Figs. 6 and 7, respectively. For each independent data set the best fits were obtained for the two-component model. In the case of native HLL lipase the maximum of fluorescence emission of the iodide-quenchable component lies at 338 nm and the maximum of the inaccessible component is at 330 nm.

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400

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I

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I

I

320

340

360

380

400

Wavelength [ nm ] Fig. 6. Fluorescence-quenching-resolved-spectraof native H. lanuginosa lipase obtained with excitation at 295 urn, using potassium iodide as the quencher: ( • ) quenchablecomponent( A,,~x= 338 nm) ; ( • ) unquenchable component (Amax= 330 nm); ( • ) total steady-stateemission spectrumof HLLlipase (Amax= 340 nm). The lowerpanel showsthe dependenceof the Stern-Volmerdynamicconstants on emissionwavelength.

400 ,

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=,

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300

320

340

360

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360

380

400

2

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380

400

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Wavelength [ nm ]

Fig. 7. Fluorescence-quenching-resolved-spectra of inhibited1-1.lanuginosa lipase obtainedwith excitationat 295 nm, usingpotassiumiodideas the quencher:(O) quenchablecomponent(Amax= 342nm); (•) unquenchable component()tmax= 342 nm); (•) total steady-stateemissionspectrumof HLLlipase(Zma,t= 340 nm). The lowerpanelshowsthe dependenceof the Stern-Volmerdynamicconstantson emissionwavelength. The spectral contributions (calculated from the areas under each resolved spectrum) of the components mentioned above to the total fluorescence spectrum were 66 and 34%, respectively. For inhibited HLL lipase the iodide-quenchable component contributes about 49% to the total fluorescence emission. The maxima of the accessible and inaccessible components lie at 342 and 334 nm, respectively. Our quenching studies have contributed to the understanding of the heterogeneous fluorescence emission from H. lanuginosa lipasel The results obtained in acrylamide-quenching experiments indicate that all four tryptophan residues within the protein are accessible to the quencher. Contrary to acrylamide, potassium iodide was found to be an effective and selective quencher for Trp89 residue. Using the FQRS method we have shown that the maxima of the Trp89 resolved emission spectra equal 338 and 342 nm within the native and inhibited forms of H. lanuginosa lipase, respectively. The spectrum of the iodide-unquenchable component, corresponding to the remaining three out of four tryptophan residues, ranges from 330 to 334 nm. This result is consistent with the maximum of the steady-state fluorescence emission spectrum obtained for the mutated form of protein. The steady-state fluorescence quenching studies of native and inhibited H. lanuginosa lipase with potassium iodide show that two different conformations of the enzyme, caused by

102

A. Stobiecka et al. / Journal of Photochemistry and Photobiology B: Biology 45 (1998) 95-102

limited movement of the short a-helical 'lid', can be monitored using the fluorescence properties of Trp89 residue.

[15]

Acknowledgements

[16]

We wish to express our thanks to Novo Nordisk for the gift of H. lanuginosa samples. We are also very grateful to Professor Zygmunt Wasylewski for supplying us with the FQRS computer program.

[17]

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