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Excited state proton transfer in 2-naphthol derivatives bound to selected sites of proteins
J, Photochem. Photobiol. A: Chem., 69 (1992) 57-66
57
Excited state proton transfer in Z-naphthol derivatives bound to selected sites of proteins A. Jankowski
and
P. Stefanowicz
Institute of Chemisby, Univmi~ of Wrvchnv, F. Joliot-Curie 14, 50-383 Wmdaw (Poland)
P. Dobtyszycki lnstitite of Organic and Physical Chemistry, Technical University, Wybrteze wspianskiego 27, SO-370 WroCaw (Poland) (Received June 20, 1991; accepted June 18, 1992)
Abstract Derivatives of Z-naphthol-6-sulphonic acid were anchored by covalent bonding to the active site of papain and to the surface of bovine serum albumin. The rate constants of excited state proton transfer for the introduced fluorophoric groups were determined by steady state fluorescence spectroscopy and compared with the data for low-molecular-weight analogues of 2-naphthol. In the light of these results and other data published previously, it is concluded that the fluorophores bound inside macromolecules are, in most cases, characterized by a higher rate of excited state proton transfer than groups situated on the surface. The unique physicochemical properties of the enzyme active site are taken into consideration in the case of the papain conjugate.
1. Introduction Proton transfer reactions play an important role in biological phenomena such as energy conversion, enzymatic catalysis, vision and photosynthesis [l--S]. The mechanism of proton transfer for reactants in solution is relatively well understood [6]. However, for biological processes proton transfer takes place in macromolecular systems, such as proteins and membranes, in which the course of the reaction may be influenced by the environment in a specific manner. The object of this work is to determine the influence of the macromolecular environment on proton transfer reactions. We investigated fluorometrically the excited state proton transfer in Auorophores bound to selected sites of proteins. It is assumed that the ground state proton transfer, which may be intrinsically different from the excited state process, shows a similar dependence on the microenvironment of the proton donor. Weak organic acids, such as 2-naphthol and its derivatives, which are characterized by lower pc values in the first singlet excited state than in the ground state, undergo very rapid proton transfer when excited ROH*+A
I&, y RO-*+AH+
lOlO-6030/92/$5.00
(I)
where A is a proton acceptor, in most cases H,O. This process is manifested by the appearance of two fluorescence bands due to ROH* and RO-* forms and a characteristic time dependence of the emission intensity on a nanosecond time scale. The kinetics of the process may be investigated by steady state or time-resolved fluorescence spectroscopy. We used 2-naphthol derivatives covalently bound to proteins for the determination of the rate of proton transfer at the binding sites. The rates of long-distance proton transfer in proteins, biological membranes and artificial analogues have been extensively studied [7-lo]. Some investigations of excited state proton transfer in macromolecules have been published in which exposure of the fluorophores to the solvent was also taken into consideration [U-13]. Recently, we have obtained derivatives of 2-naphthold-sulphonic acid (NSOH) which are suitable for modification of proteins, allowing more selective labelling than described previously [12]. NSOH groups were anchored to the active site of papain (PAP-NSOH) and to the surface of bovine serum albumin (BSA-NSOH(S)). Rate constants of excited state proton transfer were measured by steady state fluorescence spectroscopy according to the method of Weller [14]. The binding sites were characterized by determination of the accessibility
0 1992 - Elaevier Sequoia. All rights reserved
A. Jankowski et al. / Proton
58
transfer in 2-naphlhols bound IOproreins
of the fluorescent probes to the solvent. In addition, low-molecular-weight analogues of NSOH groups bound to proteins (2-naphthol-6-sulphonamides) were synthesized and investigated. The results were compared with the data for NSOH-protein conjugates. Conclusions concerning the influence of the macromolecular environment on the mechanism of proton transfer in macromolecular systems are presented.
2. Experimental
details
2.1. Synthesis and characterization
of 2-naphthol derivatives used for label&g of proteins 2-Acetyloxynaphthalene-6-sulphochloride
(NOAcSCI) was synthesized as described previously [12]. 2-Hydroxynaphthalene-6-sulphonamidoN{ethyleno)-N-iodoacetamide (JAE-NSOH) was synthesized by the reaction of NOAcSCl with a fivefold molar excess of ethylenediamine at -20 “C. The monosubstituted product was coupled with iodoacetic acid in the presence of dicyclohexylcarbodiimide in methylene chloride-dimethylformamide (1:l) at -30 “C. The product was crystallized from ethyl acetate. Elemental analysis: calculated: 38.7% C, 6.4% N, 7.4% S, 3.4% H; found: 38.6% C, 6.1% N, 7.4% S, 3.4% H. IR (KBr): 1660, 1620, 1320, 1148 cm-l. Melting point (m.p.>, 159-162 “C. The succinimide ester of Z-acetyloxynaphthalene-6-sulphonic acid (NOACS-SUCC) was synthesized by the reaction of equimolar amounts of NOAcSCl with N-hydroxysuccinimide dissolved in tetrahydrofuran in the presence of triethylamine. After evaporation of the solvent the material was treated with water. The product was crystallized from ethyl acetate. Elemental analysis: calculated: 52.9% C, 3.8% N, 8.8% S, 3.6% H; found: 52.7% C, 4.0% N, 9.5% S, 3.4% H. IR (KBr): 1760, 1745, 1630, 1390, +1185 cm-‘. M.p. 201-202 “C. 2.2. Synthesis of 2-hydroxynaphthalene-(dodecylo)Gsulphonamide dodecylsulphate
- a probe for labelling sodium micelles
2-Hydroxynaphthalene-(dodecylo)-6-sulphonamide (DA-NSOH) was prepared by the reaction of an equimolar amount of NOAcSCl with dodecylamine in the presence of a stoichiometric amount of triethylamine in methylene chloride. The acetyl blocking group was removed by hydrolysis with a large excess of NaOH in methanol solution for 3 h at room temperature. Elemental analysis: calculated: 67.5% C, 3.5% N, 8.2% S,
8.4%
H; found: 66.7% C, 3.9% N, 8.3% S, 8.5% H. IR (KBr): 1625, 1305, 1145 cm-‘. M.p. 118 “C. 2.3. Synthesis and characterization molecular-weight analogues group bound to proteins
of lowof the fluorophore
The 6-hydroxynaphthalene-2-sulphonamide of a-L-phenylalanine (Phe-NSOH) was synthesized by the reaction of NOAcSCl dissolved in dioxane-diethyl ether (1:l) with a small excess of (YL-phenylalanine dissolved in water (fivefold molar excess of NaOH). The organic solvents were removed by evaporation and the water solution was acidified to pH 1. The precipitate was crystallized from water. Elemental analysis: calculated: 61.5% C, 3.8% N, 8.7% S, 4.6% H; found: 60.9% C, 3.5% N, 8.9% S, 4.5% H; IR (KBr): 1675, 1625, 1330, 1158 cm-l. M.p. 115-118 “C. The 6-hydroxynaphthalene-Zsulphonamide of glycine (Gly-NSOH) and 6-hydroxynaphthalene-2sulphonamide (NH,-NSOH) were synthesized in a similar manner to Phe-NSOH. The 6-hydroxynaphthalene-Zsulphonamide of Eamino-capronic acid (EACA-NSOH) was prepared by the reaction of NOAcSCl with an equimolar amount of the methyl ester of l -amino-capronic acid hydrochloride in methylene chloride in the presence of a stoichiometric amount of triethylamine. The product was hydrolysed by a water solution of NaOH (fivefold molar excess) and crystallized from water. Elemental analysis: calculated: 55.9% C, 4.15% N, 9.4% S, 4.5% H; found: 55.9% C, 4.19% N, 10.3% S, 5.0% H. IR (KBr): 1708,1625, 1318, 1148 cm-‘. M.p. 151-152 “C. 2.4. Labelling of proteins The procedure for the labelling of proteins by NOAcSCl has been described elsewhere [12]. Bovine serum albumin (BSA, fraction V, Sigma) was coupled with NOAcS-Succ in the following manner. BSA (280 mg) dissolved in 4.5 ml of phosphate buffer (pH 7.5) in the presence of Triton X-100 was treated with 31 mg of NOAcS-Succ dissolved in 0.6 ml of dimethylformamide. The reagent was added dropwise with stirring. The addition of the reagent was stopped when marked precipitation in the stirred solution was noticed. After 48 h of stirring at room temperature the labelled protein was treated with 1 M hydroxylamine in neutral water solution. Removal of the acetyl blocking group from the marker bound to the protein was verified as described previously [12]. After 12 h the reaction mixture was applied
A. Jankowski et al. / Proton transfer in Z-naphthols bound to proreins
to a Sephadex G-25 coarse column (3 cmXS0 cm) equilibrated with 0.01 M of ammonium carbonate. The elution by the same buffer was monitored spectrophotometrically at 280 and 320 nm. BSA labelled by NSOH groups (BSA-NSOH(S)) was lyophilized. Papain (water soluble, Merck) was purified on a Sephadex G-75 (3 cmX7S cm) column before use. The fraction of lowest elution volume was lyophilized and used for labelling. Purified papain (PAP) (100 mg) dissolved in 3.5 ml Tris-HCl buffer (pH 7.5) containing 2 mg ethylenediaminetetraacetic acid (EDTA) was treated with 10 mg of JAE-NSOH dissolved in 0.5 ml of dioxane. The reagent was added dropwise with stirring at 5-10 “C. The reaction mixture was stirred for 3 h in the dark at room temperature. Excess reagents were removed on a Sephadex G-25 column and Uabelled protein was rechromatographed on a Sephadex G-75 column (elution by 0.01 M of ammonium carbonate). Removal of the acetyl blocking group - performed at the stage of preparation of JAENSOH - was verified as described previously [12]. The labelled PAP sample (PAP-NSOH) was lyophilized and its homogeneity checked by polyacrylamide gel electrophoresis (pH 8.5). The enmatic activity of PAP-NSOH was not tested, but t may be supposed (see below) that this modir cation yielded inactive enzyme. 13.5. Methods The amount of free SH groups in labelled and nnlabelled PAP was determined spectrophotoetrically by the method of Elhnan [15] E~,~= 13 600 M-’ cm-‘). I” The amount of marker bound per protein molecule was determined by the spectrophotometric . 16 (~~~~=4000 mM-’ err-‘). measurements were pere&man 5240 or a Specord ISS) instrument. Absorption specPhe-NSOH at pH 6, 10 and 12, recorded he Beckman apparatus, were resolved into bands by the method of Powell (apparent) of ground state pK, graphically from the spectroration curves. The error of the iaations was estimated from the maximum tion of the results in the measurement series. ues of pc in the excited state were ded from the kinetic measurements described as a ratio of the rate constants (K= or from the FGrster cycle [14]. rofluorometric measurements were performed using a Perkin-Elmer model 204
59
apparatus supplied with a Perkin-Elmer 56 recorder. Spectrofluorometric titrations were performed as described elsewhere [12]. pH values were adjusted by addition of HCl or NaOH. At low pH (O-l) some quenching of NSOH fluorescence was apparent. True fluorescence intensities (F) were calculated by taking into account the isoemissive points visible in the fluorescence spectra of NSOH conjugates titrated at lower acid concentration. Thus eqn. (2) was applied F0 = F&,(FCI, isalFiscJ
(2)
intensity at a given where Fobs is the fluorescence (low) pH value observed at a given wavelength, Fo,im is the fluorescence intensity at neutral pH (no quenching by Cl- or H+ ions) at the isoemissive wavelength, i.e. the wavelength at which the emission intensity of the ROH form is equa1 to that of the RO-* form (this wavelength is determined as the point at which the fluorescence spectra of a given NSOH derivative taken at various pH values intersect) and Fi, is fluorescence intensity at a given (low) pH value at the isoemissive wavelength. Excited state proton transfer rate constants were obtained from the spectrofluorometric titration results by the method of Weller [14]. Equations (3) and (4) were used (see also eqn. (1))
(4) Thus & was obtained from eqn. (3) where T,, is the lifetime of the first singlet excited state of the ROH* form (see eqn. (l)), & is the fluorescence quantum yield of the ROH form at neutral pH (see Fig_ 2, Section 3) determined by the relative method [18] (see below) and &, and & are the fluorescence quantum yields of pure ROH* and RO-* forms determined by the relative method [18] using, as standard, Schaefer’s acid characterized by emission band positions very similar to those _pf our samples, with &=0.24 and &=0.49 [12]. kpt was obtained by fitting the experimental results to eqn. (4) as a linear dependence of (+/ &,)l,(@l&) on F[H+]. The slope of this pl_ot s =k,,~&,~~,,, with Q and $, measured and k,, determined from eqn. (3), gives the value of kpt. In eqn. (4) ~b is the lifetime of the RO-* form (see eqn. (1)) and F=z~z~(~.O~JO-~)/(~+~~‘) where J is the ionic strength, z1 and z, are the charges of the reactants and 4 and 4’ are the fluorescence quantum yields at a given pH value
60
A. Jankowski
et al. / Proton transfer
of the ROH* and RO-* fluorophore forms respectively. As opposed to thevalue of 4, occurring in eqn. (3) the values 4 and $’ from eqn. (4) should be measured at acidity values sufficiently high to obtain significant changes in the fluorescence spectra with pH (see Fig. 2, Section 3). The lifetime measurements of the first singlet excited state in the compounds studied were performed using an impulse fluorometer LIF 200 (GDR) with a laser source IGT 50 (GDR) (N, u,,,=337 nm). The fluorescence (4 decays were fitted to a single, double or triple exponential (5) The rate constants of dynamic fluorescence quenching (k,) by acrylamide for NSOH groups bound to BSA or Phe were determined from the linear Stern-Volmer plots. The fluorophores bound to proteins may be buried inside the hydrophobic core of the macromolecule and thus may, in some part, be inaccessible to the solvent within the lifetime of the excited state. In this case no linear Stern-Volmer dependence of the fluorescence intensity on the concentration of the ionic quencher is obtained. A modified Stern-Volmer equation enables the fraction of fluorophores accessible to the quencher (fr) to be determined [19]. This value is a measure of the accessibility of the fluorophores to the polar solvent and was employed to charactcrize the position of the sites of binding of NSOH groups in macromolecules using the method of Lehrer [19].
3. Results PAP preparations contain isoenzymes [ZO]. In the chromatographic elution profile of the commercial PAP used in this study three peaks were visible. Only the first, well-resolved fraction of the lowest elution volume was used for modification with JAE-NSOH. The sample of labelled PAP (PAP-NSOH) obtained was homogeneous in polyacrylamide gel electrophoresis. PAP contains one free SH group (cysteine (Cys) 25) situated in the active site [21]. The amount of SH groups in purified PAP decreased after labelling (Table 1) by a quantity corresponding to the amount of bound marker, which was determined independently by a spectrophotometric method. It was concluded that in PAP-NSOH the enzyme was labelled selectively at the SH group
in 2-naphthols
bound
to proteins
positioned in the catalytic niche. However, it should be noted that a small quantity (15%) of PAP remained unlabelled (see Table 1). In the case of BSA-NSOH(S) the protein was labelled using NOAcS-Succ which is expected, in analogy with carbonyl succinimide esters [22, 231, to be more selective than the NOAcSQ used previously [12] towards the amino groups of the protein. It should be noted, however, that our results suggest a possible formation of unstable side products in the reaction of NOAcS-Succ with proteins. The substitution by the fluorescent marker was 0.85 group per protein molecule in PAP-NSOH and 0.7 group per protein molecule in BSANSOH(S) (Table 1). The fluorescence of tryptophan in PAP-NSOH (excited at 280 nm) was identical with that of the unmodified enzyme, suggesting that the native structure of the protein was not disturbed. The same holds for other modified proteins mentioned in this work with the exception of BSA-NSOH(S) where tryptophyl emission is markedly decreased compared with that of the native protein. The fraction of fluorophores accessible to the ionic quencher (JJ) in labelled proteins is given in Table 2. This value is a measure of the exposure of the fluorophoric groups to the solvent. It is evident that in BSA-NSOH(S) the fluorophores are bound preferentially on the surface of the protein molecule and in PAP-NSOH they are located in the interior. The fluorescence spectra and fluorometric titration curves of PAP-NSOH and BSA-NSOH(S) are presented in Figs. 1 and 2 respectively. In Fig. 1 the higher energy band (h,,,.,=363 nm) is due to the ROH* fluorophore form and the lower energy band (A,,,,,= 445 nm) to the RO-* form. The titration curve for BSA-NSOH(S) in analogy with those of other labelled proteins studied previously [12], shows no marked changes in the ROH” and RO-* fluorescence bands at pH 3-7 and both emission bands are observed at pH 1-12. For PAPNSOH, at pH 6-6.5 there is a very rapid rise of the RO-* form emission intensity and at pH 7 only the long-wavelength fluorescence band is observed experimentally. This behaviour is probably caused by deprotonation of the histidine (His) residue positioned close to the active site [24]. Negative logarithms of the equilibrium constants of the proton transfer reaction in the phenolic group (eqn. (1)) of the investigated compounds in the ground (pK,) and excited (pK,*) states are given in Table 3. The ground state pK, values are similar within experimental error ( f 0.2) for most
A. Jankowski et al. I Proton transfer in 2-naphfhok bound to proteins TABLE
1. The amount
of free SH
61
groups and bound marker per protein molecule in PAP and PAP-NSOH
Compound
SH” (M dcm-3,
PAPb (M dcm+)
[NSW (M dcm-‘)
PAP (unlabelled) PAP-NSOH
1.32 x lo-’ 2.96 x10-s
1.37x 1o-5 1.93x 1o-5
0 1.79x10-5
[NSOH]/[PAP]
0.96 0.15
0 0.93
*Q~= 13 600 mM_’ cm-’ was assumed. ‘From gravimetric data. determined from c3353 =4000 r&i-’ cm-l. dJ&perimental error, 5%.
TABLE 2. Accessibility to ionic quencher VI) and fluorescence quantum yields (&, &,) and lifetimes (TV 4$ for NSOH groups bound to proteins and for Phe-NSOH (unprimed symbols for ROH* form and primed symbols for RO-* form) Compound
fi
4
&I
% (ns)
7; (us)
PAP-NSOH BSA-NSOH(S) BSA-NSOHQ) Phe-NSOH
0.32 0.74 0.30
0.048 0.114 0.03 0.25
0.102 0.17 0.13 0.5
3.41 5.2 0.84 7.17
6.73 1155 2.9 10.23
Fig. 2. Spectrofluorometric titration and BSA-NSOH (broken line).
Fig. 1. Fluorescence spectra at pH 6: (1) PAP-NSOH (excitation at 320 nm; concentration, 3 X 10e6 M); (2) BSA-NSOH(S) (excitation at 320 nm; concentration, 3 x lo-” M).
cases. A higher value is obtained only for NSOH groups bound to sodium dodecylsulphate (SDS) micelles where the negative charge density of the micelles explains the effect observed. The excited state pK,” values of the NSOH derivatives differ significantly depending on whether position 6 of the naphthalene ring is substituted by the sulphonamide group. This may be caused by a contribution from the structures with a negative charge density localized at position 6 of the naphthalene ring [27]. Such structures probably contribute more to the excited state than to the ground state wavefunction because of the
of PAP-NSOH
(full line)
greater extent of negative charge migration from the phenolic oxygen to the ring in the excited state. The localization of electrons in position 6 of the naphthalene ring must be hindered for derivatives substituted in this position by a group bearing a negative charge. The opposite effect is expected for compounds substituted at position 6 by an electron-withdrawing group. This may explain the larger decrease in the pK,* values in the 2naphthol derivatives substituted in position 6 by sulphonamide, an electron-withdrawing group, than in analogues bearing a negative charge (Schaefer’s acid). The similar pK,* values for proteinNSOH conjugates and low-molecular-weight NSOH-sulphonamides (Table 3) confirm the view that the NSOH groups in our conjugates are bound to proteins by means of the sulphonamide bond. The interpretation presented above is in agreement with the effects of deprotonation of the sulphonamide nitrogen atom at pH 10-12 on the absorption and fluorescence spectra of Phe-NSOH and other investigated compounds (Table 4). The_ rate con_stants of excited state proton transfer (k,, and kpt, see eqn. (1)) for the NSOH-
A. lankowski
62
et al. I Proton
transferin
2-naphthols bound to proteins
TABLE 3. Negative logarithms of the equilibrium constants of the prcton transfer reactions (eqn. (1)) in the ground excited (pK:) states and rate constants for the excited state reaction (kpt and kpt, see eqn. (1)) Compound
5, (s-’
PK e
(PK.) and
PJ? (Farster
M-’ 1”x lo+?)
cycle) ‘2.Naphthol” Schaefer’s acidb Phe-NSOH NH,-NSOH EACA-NSOH Gly-NSOH SDS-NSOH PAP-NSOH BSA-NSOH(S) BSA-NSOH(1) ‘Data from ref. 25. bData from ref. 26. ‘Experimental error,
9.46 9.1 9.2 8.9 9.2 9.1 11.05 8.7 9.3 9.7
a.72 8.5 7.92 9.98 0.27 8.17 1.44 0.82
Absorption
Solution
Hz0 (PH 6) 0.1 n KOH (pH 10) 1 n KOH (pH 13) investigated
2.5 1.6 0.6 0.7
0.6
* 0.2.
TABLE 4. Effect of ionization of the sulphonamide of Phe-NSOH”, resolved into gaussian components,
“Other
2.8 1.66 0.51 0.7 0.48 0.49 3.90 0.48 1.14 0.32
694 406 39.6 56.1 33.6 43.3 3900 72.8 38.5 18.7
1.1 10.2 12.1 * 1 11.2*2 11.1*1 13.9*1 0.4*1 23.9+2 2.7 * 1 10.4
sulphonamides
nitrogen atom on the positions of the band maxima in the absorption and in the fluorescence spectrum spectrum
Fluorescence spectrum (RO-*)
maxima
(nm)
‘I-% (nm)
$n)
$)
298.4 344.1 348.4
279.6 305.7 301.1
236.4 246.0 246.2
show similar effects but the spectra
conj_ugates studied are given in Table 3. The error of k,, determination (given in Table 3) was estimated from the maximum deviation in the fluorometric measurements (intensity-and lifetime}. The error of determination of k,, was higher because of the necessary addition of HCl (see Section 2.5). This error has not been estimated. For the low-molecular-weight sulphona_mides studied, the deprotonation rate constants k,, are practically identical within experimental error. For the NSOH groups bound to the surface of the protein in BSA-NSOH(S), the kpl value is much lower and, for NSOH groups in the catalytic site of PAP-NSOH, it is higher than for the lowmolecular-weight analogues. In protein-NSOH conjugates the fluorophoric groups are shielded, to a large extent, from the solvent by neighbouring groups of the macromolecule. This implies a decrease in the steric accessibility for the proton transfer reaction to the solvent for the groups bound to a protein. If this reaction were diffusion controlled, a higher reaction rate would be expected for sites of better accessibility to the solvent, such as those in BSA-
spectrum
were not resolved
into separate
445 443 436 components.
NSOH(S), than for PAP-NSOH where the accessibility of the fluorophore is much lower (Table 2). The present results suggest that, for fluorophores buried inside a protein interior, e.g. in PAPNSOH, proton transfer through preformed hydrogen bonds takes place. To verify the dependence of the proton transfer rate on the accessibility of the proton donor to the solvent, we studied the dependence of the excited state proton transfer in NSOH-protein conjugates on the rate of water diffusion by another method. An increase in protein concentration above a certain limit causes aggregation of the macromolecules. This will lead to a decrease in the probability of encounter of water molecules with the excited fluorophores. If this effect influences the rate of proton transfer, an increasing protein concentration should cause definite changes in the fluorescence spectra. To test the influence of water diffusion on the proton transfer reaction, we investigated the effect of an addition of unlabelled protein on the fluorescence of prokin-NSOII conjugates (Fig. 3). In this experiment the influence of increasing sample turbidity on the
A. Jankowski e[ al. I Proton
transferin 2-naphtholr bound to proteins
63
centration is nearly linear, as for low-molecularweight sulphonamides. Therefore a Stern-Vohner plot can be used in both cases. To compare the exposure of fluorophores in BSA-NSOH(S) with those in low-molecular-weight NSOH-sulphonamides we used the fluorescence quenching wnstants by acrylamide (k,) given in Table 5 k, = K&o 02
_
2 5 ,
100
x I‘
I
I
. ‘200
!
!
s
PROTEIN Fig. 3. Dependence of Q at the peak maxima tonated (RO-*) forms (1) BSA-NSOH(S); (2)
1.
’ ‘330
:I
I
a
::
Fwmll
,
CONCENTRATION
the ratio of the fluorescence intensities of the protonated (ROH*) and deproan the addition of unlabelled protein: PAP-NSOH.
fluorescence spectra was eliminated by subtracting from the spectra the baseline obtained by inserting in the exciting light beam a solution of unlabelled BSA of equal concentration as the sample investigated. The unlabelled BSA shows no absorbance at the exciting wavelength (320 nm). It can be seen from Fig. 3 that PAP-NSOH fluorescence is insensitive to a rise in protein concentration. However, BSA-NSOH(S) shows a marked enhancement of the fluorescence intensity (F) of the protonated form (ROH*) and a decrease in emission from the deprotonated form (RO-*) with increasing protein concentration. This is illustrated in Fig. 3 by an increase in the ratio F ROHsIFRO-*with increasing protein concentration. We conclude that the excited state proton transfer reaction in BSA-NSOH(S), but not in PAP-NSOH, is sensitive to the rate of diffusion of water towards the excited fluorophores. Thus a decrease in the diffusion coefficient for water causes a decrease in the rate of excited state proton transfer in this sample. Thus a reduction in the rate of excited state proton transfer in BSA-NSOH(S) compared with the value for low-molecular-weight sulphonamides (Table 3) must be at least partly due to the decrease in the accessibility of the Uuorophores bound to the protein surface. A comparison of the exposure of the fluorophores bound to BSA with those in lowermolecular-weight sulphonamides cannot be performed by the method of Lehrer [19] (see Section 2.5). However, because the fraction of fluorophores accessible to the ionic quencher (fi) in BSANSOH(S) is high (Table 2), the dependence of the fluorescence intensity on the quencher con-
(6)
where I&, is the Stem-Vohner constant and Q is the lifetime of the excited state of the pure ROH* form. From the results in Table 5 we conclude that the 50% decrease in the accessibility of NSOH groups in BSA-NSOH(S) with respect to the value for Phe-NSOH (and other low-molecular-weight analogues) canno_t fully account for the 70% reduction in the kpf value (Table 3). The determination of the reason for the reduction in the proton transfer rate for BSA-NSOH(S) requires further investigation. It was of inte_rest to determine whether the dependence of k,, on pKf for the investigated compounds (Table 3) would yield a similar fiicture to that of many other reactions of organic (acids, including the excited state data for 2-naphthol analogues [6, 281. The relationship between log k,, and log K,* for the compounds studied (Fig. 4) shows that the data for low-molecular-weight NSOH-sulphonamides can be fitted to the curve for other 2-naphthol derivatives of low molecular weight. In this way a plot very similar to that published previously [28, 291 is obtained. This suggests that the excited state proton transfer in low-molecular-weight analogues follows the same mechanism, and is characterized by parameters such as the proton vibrational strength in the initial and final states of the reaction, the energy of medium reorganization, the proton tunnelling probability, etc. [29, 301. The data for the majority of macromoleculeNSOH conjugates (NSOH-protein and NSOHSDS) cannot be fitted to the plot in Fig. 4. Only the result for PAP-NSOH (11, Fig. 4) fits the curve constructed for low-molecular-weight analogues of 2-naphthol. This is unexpected because TABLE 5. Rate constants (k.J by acrylamide
of dynamic
fluorescence
quenching
Compound
J&v
70 w
kqx lo-’
Phe-NSOH BSA-NSOH(S)
2.00 0.745
7.17 5.20
2.78 1.43
Symbols are explained
in the text.
64
A. lankowski
et al. I Proton
transferin 2-naphth&
Fig. 4. Dependence of log &, on log K: (Breinstedrelation): (1) 2-naphthol; (2) 1-naphthol; (3) 2-naphthol-6.sulphonate; (4) PheNSOH; (5) Gly-NSOH; (6) SDS-NSOH, (7) 7-methyl-2.naphthol; (8) 6.methyl-2.naphthob (9) 1.chloro-Z-naphthol; (10) l-bromo2-naphthol; (11) PAP-NSOH; (12) BSA-NSOH(I); (13) BSANSOH(S); (14) BSA-NSOH(A). Filled circles, low-molecularweight derivatives of Z-naphthol; open circles, macromoleculeNSOH conjugates. Data for samples t-3 were obtained from refs. 25 and 26, for samples 4-6 and 11-14 from our results and for samples 7-10 from ref. 28. Samples 12 and 14 are described in detail elsewhere [12].
of the position of the NSOH group in the catalytic site in PAP-NSOH. This problem requires further study. From the data in Fig. 4 it can be concluded that the majority of macromolecule-NSOH conjugates are characterized by a different mechanism of excited state proton transfer to the low-molecular-weight analogues.
4. Discussion In this work we studied the rates of excited state proton transfer in fluorophores bound selectively in the active site of PAP in PAP-NSOH. For the other sample studied (BSA-NSOH(S)) the position of the proton donor was less precisely known, but it was characterized in terms of the exposure of the fluorescent groups to the solvent. Some results for other NSOH-protein conjugates obtained previously [12] were used for comparison. The high rate of proton transfer for groups of low accessibility to water (Table 3) is most probably due to the mechanism of proton transfer through intramolecular hydrogen bonds. Chains of hydrogen bonds may be formed by side groups of some amino acids or by the peptide backbone [31, 321. There is also a possibility of proton transfer through chains of hydrogen-bonded water molecules inside the protein macromolecule [24, 331. For proton transfer through chains of hydrogen bonds, which are more stable than those in bulk water, a very
bound to proteins
high rate and a transmission coefficient close to unity may be expected [32], Two ionogenic groups (His 159 and aspartic acid (Asp) 175), which can act as proton acceptors, are present in the active site of PAP. In unmodified PAP, Asp 175 and His 159 are probably engaged in hydrogen bonding [21]. This statement, based on crystallographic data [24], in connection with the low ground state pK, value of PAP-NSOH (Table 3), indicates that the NSOH group bound in the active site of PAP is not engaged in intramolecular hydrogen bonding to Asp 175 or His 159. The Asp 148 residue is separated by too large a distance to allow hydrogen bonding with the NSOH group bound to Cys 25. Therefore, in the case of PAP-NSOH, proton transport through internal hydrogen-bonded water molecules of the protein is more probable than transfer through the intramolecular hydrogen bonds of the protein. Proton transfer through chains of four hydrogenbonded water molecules in bulk water is also responsible for the excited state deprotonation of low-molecular-weight 2.naphthol analogues in water solution [34]. The reason for the enhancement of the rate of proton transfer for NSOH groups buried inside the protein core relative to that for low-molecular-weight analogues may be the reduction in the energy of medium reorganization (E,) [29, 30, 33-361 and/or special ordering of internal water molecules surrounded by the protein network 133, 37, 381. The results of the fluorometric titration (Fig. 2) suggest a role for the partial electrostatic charge of His 159, which is expected to release a proton at pH 6 [24, 331, in increasing the rate of proton transfer in PAPNSOH. A comparison of the &, values for PAP-NSOH and BSA-NSOH(1) (a sample described in detail elsewhere [12]) with the data on solvent accessibility (Table 2) shows that the degree of enhancement of the proton transfer rate for fluorophores situated in the hydrophobic core of proteins is different for groups of comparable solvent accessibility. We suppose that, in BSANSOH(1) (see ref. 12), where the labelling was non-homogeneous but the NSOH groups were preferentially located inside the hydrophobic core, proton transfer through the intramolecular hydrogen bonds of the protein was significant. In contrast, the k,, values for BSA-NSOH(S) and other conjugates characterized by highly accessible NSOH groups are similar (see Tables 2 and 3 and table 2, ref. 12). For NSOH groups bound on the surface of macromolecules some immobilization of water at the interface boundary
A. Jankowski et al. I Pmton transfer in 2-naphtholrr bound to proteins
may influence proton transfer [lo, 331. Because of the non-zero surface charge of the protein molecule the probability of geminal recombination of the proton immediately after its release from the excited NSOH group must be taken into account [lo]. Both of these effects (immobilization of water and geminal recombination) may be responsible for the decrease in the proton transfer rate for NSOH groups bound to the surface of proteins compared with the value for the lowmolecular-weight analogues. We were unable to estimate experimentally the contribution of geminal recombination in the fluorescence spectra of our samples, but it can be supposed that its role is not very significant since the pI(,* values determined from the Flirster cycle are roughly coincident with the data obtained from our kinetic measurements (Table 3). Gutman et al. [7, lo] investigated proton transfer reactions in chromophores bound to macromolecular systems such as proteins, micelles and membranes. These workers obtained, for proteinbound flyorophores, higher protonation rate constants (k,f), and comparable deprotonation rate constants (k,,) with our results for surface-bound NSOH groups. The position of the proton donor and acceptor in terms of the accessibility to the solvent was not specified in the work of Gutman et al. [7, IO].
biological phenomena, further experimental evidence on the mechanism of proton transfer in biological systems is required. Acknowledgments This work was supported by a grant from the Committee of Scientific Research (Z-0760-91-01) and by the Technical University of Wroclaw. References 1 P. Bayer, B. Chance,
2 3 4 5 6 7 8 9 10 11
12
5. Conclusions The following conclusions may be drawn from our results. (1) The rate of proton transfer for groups located in the hydrophobic core of proteins is enhanced relative to that for low-molecular-weight analogues. This conclusion confirms a similar opinion reached previously 111-131. (2) The enhancement of the reaction rate for internally bound NSOH groups is probably due to proton transfer via the intramolecular hydrogen bonds of the protein or via chains of internal, structural water molecules. (3) A decrease in the rate of proton transfer for surface-bound groups compared with low-molecular-weight analogues is due mainly, but not exclusively, to a reduction in the accessibility to water. It has been postulated on theoretical grounds [36] that the enhancement of the rate of proton transfer inside protein molecules is of major importance in enzymatic Catalysis. As proton transfer reactions also play an important role in other
6.5
13 14 15 16 17 18 19 20 21 22 23 24 2.5 26 27 28
29
L. Emster, P. Mitchell, E. Racker and E. Slater, Annu. Rev, B&hem., 46 (1977) 955-1026. H. Panefsky, J. Biol. Chem., 26 (1985) 13 735-13 741. P. Dupuis and M. El Sayed, Can. J. Chem., 63 (1985) 169%1704. G. MaImstrom, FEBS Let&, 250 (1989) 9-21. R. Kraayenhoff, F. De Wolf, H. van Wairaven and K. Krab, Bioe~ectrochem. Bioeng., 16 (1986) 273-285. R. P. Bell, The Proton in Chemistry, Chapman and Hall, London, 1973, p_ 250. M. Gutman, E. Nachliel and E. Gershon, Biochemisv, 24 (1985) 2937-2941. M. Prats, J. F. Tocanne and J. Teissie, Eur. J. Biochem., 162 (1987) 379-385. L. Keszthelyi,Retinal Proteins, Science Press, Utrecht, 1987, pp. 241-250. M. Gutman and E. Nachliel, Biochim. Biophys. Acta, 1015 (1990) 391-414. P.-S. Song, Proc. Int. Symp. on Photobiology and Biotechnology, Po.znan, 1989, Politechn., 85-99, Poznan Technical University, 1989. A. Jankowski and P. Stefanowicz, Stud. Biophys., I31 (1989) 5566. N. Chattopadhyay, T. Chakrabarthy, A. Nag and M. Chowdhury, J. Photochem. PhotobioL A: Chem., 52 (1990) 19%204. A. Weller, in E. Porter and B. Stevens (eds.),Progrw Reaction Kinetics, Vol. 1, Pergamon, Oxford, 1961, pp. 187-214. M. Ellman, Arch. Biochem. Biophys., 82 (1959) 7&75. H. JatFe and M. Orchin, Theory and Applications of W Soectrosco~ Wiley, New York, 1965, p. 565. M. Powell, Comput. I., 7 (1965) 303-310. D. Eaton, _7.Photochem. Phorobiol. B: BioZ., 2 (1988) 523-530. S. Lehrer, Biochemirtry, IO (1971) 3254-3262, Th. Boiler, in M. Dalling led.), Plant Proteolytic Enzymes, Vol. 1, CRC Press, Coca Raton, FL, 1986, pp. 67-96. C. Ryan and U. Walker-Simons, The Biochemistry of Plants, Vol. 6, Academic Press, New York, 1981, pp. 321-350. G. Means and R. Feeney, Chemical Modijication of Proteins, Holden Day, San Francisco, 1971, p. 97. I. Becker and M. Wilchek, B&him Biophys. Acra, 264 (1972) 165. I. Kamphuis, J. Drength and E. Balus, .I Mol. BioZ., I82 (1985) 317-329. A. Weller, 2. Phys. Chem. NF, I7 (1958) 22&245. 5. Clark, S. Shapiro, P. Campillo and K. Winn, I. Am. Chem. Sot., 101 (1979) 746748. T. Yokoyama, R. Taft and M. Kamlet, Spectrochim. Acfa, Part A, 40 (1984) 669673. I. Ireland and P. Wyatt, Adv. Phys. Orp. Chem., 12 (1976) 131-135. J. Suhnel and K. Gustav, I. P&I. Chem., 324 (1982) 71-81.
A. Jankowski et al. / Proton
66 30
framferin
and A. Kuznecov, Kinerika Chim. Reakcij v AN SSSR, Moscow, 1973, p. 170. 31 J. Nagle and H. Morowitz, Proc. Natl. Acad. Sci. USA, 75 D. Dogonadze
Po~amych (1978)
32
Rash:
298-302.
A. Bruenger,
Z. Schulten
and K. Schulten,
NF, 136 (1983) 163. 33 J. Dijkman, R. Osman and H. Weinstein, Chem., 35 (1989) 241-252.
Z. Phys. Chem. Int. J: Quuontum
34
2-naphtholv
hound
to proteins
J. Lee, G. Robinson,
S. Webb,
L. Phillips
and J. Clark, 1
Am. Chem.
Sm., 108 (1986) 6538-6542. 35 J. Suhnel, J. Phys. 0%. Chem., 3 (1990) 6268. 36 L. Krishtalik, J. Mol. C&II., 47 (1988) 211-218.
37 P. Gascoyne,
R. Pethig and A. Sent-Gyorgyi,
Sci. USA, 78 (1981) 261-265. 38 G. Careri, M. Geraci and J. Rupley,
USA, 82 (1985) 5342-5346.
tic.
Natl.Acad.
ptmz. Nurl. Acud. Sci
57
Excited state proton transfer in Z-naphthol derivatives bound to selected sites of proteins A. Jankowski
and
P. Stefanowicz
Institute of Chemisby, Univmi~ of Wrvchnv, F. Joliot-Curie 14, 50-383 Wmdaw (Poland)
P. Dobtyszycki lnstitite of Organic and Physical Chemistry, Technical University, Wybrteze wspianskiego 27, SO-370 WroCaw (Poland) (Received June 20, 1991; accepted June 18, 1992)
Abstract Derivatives of Z-naphthol-6-sulphonic acid were anchored by covalent bonding to the active site of papain and to the surface of bovine serum albumin. The rate constants of excited state proton transfer for the introduced fluorophoric groups were determined by steady state fluorescence spectroscopy and compared with the data for low-molecular-weight analogues of 2-naphthol. In the light of these results and other data published previously, it is concluded that the fluorophores bound inside macromolecules are, in most cases, characterized by a higher rate of excited state proton transfer than groups situated on the surface. The unique physicochemical properties of the enzyme active site are taken into consideration in the case of the papain conjugate.
1. Introduction Proton transfer reactions play an important role in biological phenomena such as energy conversion, enzymatic catalysis, vision and photosynthesis [l--S]. The mechanism of proton transfer for reactants in solution is relatively well understood [6]. However, for biological processes proton transfer takes place in macromolecular systems, such as proteins and membranes, in which the course of the reaction may be influenced by the environment in a specific manner. The object of this work is to determine the influence of the macromolecular environment on proton transfer reactions. We investigated fluorometrically the excited state proton transfer in Auorophores bound to selected sites of proteins. It is assumed that the ground state proton transfer, which may be intrinsically different from the excited state process, shows a similar dependence on the microenvironment of the proton donor. Weak organic acids, such as 2-naphthol and its derivatives, which are characterized by lower pc values in the first singlet excited state than in the ground state, undergo very rapid proton transfer when excited ROH*+A
I&, y RO-*+AH+
lOlO-6030/92/$5.00
(I)
where A is a proton acceptor, in most cases H,O. This process is manifested by the appearance of two fluorescence bands due to ROH* and RO-* forms and a characteristic time dependence of the emission intensity on a nanosecond time scale. The kinetics of the process may be investigated by steady state or time-resolved fluorescence spectroscopy. We used 2-naphthol derivatives covalently bound to proteins for the determination of the rate of proton transfer at the binding sites. The rates of long-distance proton transfer in proteins, biological membranes and artificial analogues have been extensively studied [7-lo]. Some investigations of excited state proton transfer in macromolecules have been published in which exposure of the fluorophores to the solvent was also taken into consideration [U-13]. Recently, we have obtained derivatives of 2-naphthold-sulphonic acid (NSOH) which are suitable for modification of proteins, allowing more selective labelling than described previously [12]. NSOH groups were anchored to the active site of papain (PAP-NSOH) and to the surface of bovine serum albumin (BSA-NSOH(S)). Rate constants of excited state proton transfer were measured by steady state fluorescence spectroscopy according to the method of Weller [14]. The binding sites were characterized by determination of the accessibility
0 1992 - Elaevier Sequoia. All rights reserved
A. Jankowski et al. / Proton
58
transfer in 2-naphlhols bound IOproreins
of the fluorescent probes to the solvent. In addition, low-molecular-weight analogues of NSOH groups bound to proteins (2-naphthol-6-sulphonamides) were synthesized and investigated. The results were compared with the data for NSOH-protein conjugates. Conclusions concerning the influence of the macromolecular environment on the mechanism of proton transfer in macromolecular systems are presented.
2. Experimental
details
2.1. Synthesis and characterization
of 2-naphthol derivatives used for label&g of proteins 2-Acetyloxynaphthalene-6-sulphochloride
(NOAcSCI) was synthesized as described previously [12]. 2-Hydroxynaphthalene-6-sulphonamidoN{ethyleno)-N-iodoacetamide (JAE-NSOH) was synthesized by the reaction of NOAcSCl with a fivefold molar excess of ethylenediamine at -20 “C. The monosubstituted product was coupled with iodoacetic acid in the presence of dicyclohexylcarbodiimide in methylene chloride-dimethylformamide (1:l) at -30 “C. The product was crystallized from ethyl acetate. Elemental analysis: calculated: 38.7% C, 6.4% N, 7.4% S, 3.4% H; found: 38.6% C, 6.1% N, 7.4% S, 3.4% H. IR (KBr): 1660, 1620, 1320, 1148 cm-l. Melting point (m.p.>, 159-162 “C. The succinimide ester of Z-acetyloxynaphthalene-6-sulphonic acid (NOACS-SUCC) was synthesized by the reaction of equimolar amounts of NOAcSCl with N-hydroxysuccinimide dissolved in tetrahydrofuran in the presence of triethylamine. After evaporation of the solvent the material was treated with water. The product was crystallized from ethyl acetate. Elemental analysis: calculated: 52.9% C, 3.8% N, 8.8% S, 3.6% H; found: 52.7% C, 4.0% N, 9.5% S, 3.4% H. IR (KBr): 1760, 1745, 1630, 1390, +1185 cm-‘. M.p. 201-202 “C. 2.2. Synthesis of 2-hydroxynaphthalene-(dodecylo)Gsulphonamide dodecylsulphate
- a probe for labelling sodium micelles
2-Hydroxynaphthalene-(dodecylo)-6-sulphonamide (DA-NSOH) was prepared by the reaction of an equimolar amount of NOAcSCl with dodecylamine in the presence of a stoichiometric amount of triethylamine in methylene chloride. The acetyl blocking group was removed by hydrolysis with a large excess of NaOH in methanol solution for 3 h at room temperature. Elemental analysis: calculated: 67.5% C, 3.5% N, 8.2% S,
8.4%
H; found: 66.7% C, 3.9% N, 8.3% S, 8.5% H. IR (KBr): 1625, 1305, 1145 cm-‘. M.p. 118 “C. 2.3. Synthesis and characterization molecular-weight analogues group bound to proteins
of lowof the fluorophore
The 6-hydroxynaphthalene-2-sulphonamide of a-L-phenylalanine (Phe-NSOH) was synthesized by the reaction of NOAcSCl dissolved in dioxane-diethyl ether (1:l) with a small excess of (YL-phenylalanine dissolved in water (fivefold molar excess of NaOH). The organic solvents were removed by evaporation and the water solution was acidified to pH 1. The precipitate was crystallized from water. Elemental analysis: calculated: 61.5% C, 3.8% N, 8.7% S, 4.6% H; found: 60.9% C, 3.5% N, 8.9% S, 4.5% H; IR (KBr): 1675, 1625, 1330, 1158 cm-l. M.p. 115-118 “C. The 6-hydroxynaphthalene-Zsulphonamide of glycine (Gly-NSOH) and 6-hydroxynaphthalene-2sulphonamide (NH,-NSOH) were synthesized in a similar manner to Phe-NSOH. The 6-hydroxynaphthalene-Zsulphonamide of Eamino-capronic acid (EACA-NSOH) was prepared by the reaction of NOAcSCl with an equimolar amount of the methyl ester of l -amino-capronic acid hydrochloride in methylene chloride in the presence of a stoichiometric amount of triethylamine. The product was hydrolysed by a water solution of NaOH (fivefold molar excess) and crystallized from water. Elemental analysis: calculated: 55.9% C, 4.15% N, 9.4% S, 4.5% H; found: 55.9% C, 4.19% N, 10.3% S, 5.0% H. IR (KBr): 1708,1625, 1318, 1148 cm-‘. M.p. 151-152 “C. 2.4. Labelling of proteins The procedure for the labelling of proteins by NOAcSCl has been described elsewhere [12]. Bovine serum albumin (BSA, fraction V, Sigma) was coupled with NOAcS-Succ in the following manner. BSA (280 mg) dissolved in 4.5 ml of phosphate buffer (pH 7.5) in the presence of Triton X-100 was treated with 31 mg of NOAcS-Succ dissolved in 0.6 ml of dimethylformamide. The reagent was added dropwise with stirring. The addition of the reagent was stopped when marked precipitation in the stirred solution was noticed. After 48 h of stirring at room temperature the labelled protein was treated with 1 M hydroxylamine in neutral water solution. Removal of the acetyl blocking group from the marker bound to the protein was verified as described previously [12]. After 12 h the reaction mixture was applied
A. Jankowski et al. / Proton transfer in Z-naphthols bound to proreins
to a Sephadex G-25 coarse column (3 cmXS0 cm) equilibrated with 0.01 M of ammonium carbonate. The elution by the same buffer was monitored spectrophotometrically at 280 and 320 nm. BSA labelled by NSOH groups (BSA-NSOH(S)) was lyophilized. Papain (water soluble, Merck) was purified on a Sephadex G-75 (3 cmX7S cm) column before use. The fraction of lowest elution volume was lyophilized and used for labelling. Purified papain (PAP) (100 mg) dissolved in 3.5 ml Tris-HCl buffer (pH 7.5) containing 2 mg ethylenediaminetetraacetic acid (EDTA) was treated with 10 mg of JAE-NSOH dissolved in 0.5 ml of dioxane. The reagent was added dropwise with stirring at 5-10 “C. The reaction mixture was stirred for 3 h in the dark at room temperature. Excess reagents were removed on a Sephadex G-25 column and Uabelled protein was rechromatographed on a Sephadex G-75 column (elution by 0.01 M of ammonium carbonate). Removal of the acetyl blocking group - performed at the stage of preparation of JAENSOH - was verified as described previously [12]. The labelled PAP sample (PAP-NSOH) was lyophilized and its homogeneity checked by polyacrylamide gel electrophoresis (pH 8.5). The enmatic activity of PAP-NSOH was not tested, but t may be supposed (see below) that this modir cation yielded inactive enzyme. 13.5. Methods The amount of free SH groups in labelled and nnlabelled PAP was determined spectrophotoetrically by the method of Elhnan [15] E~,~= 13 600 M-’ cm-‘). I” The amount of marker bound per protein molecule was determined by the spectrophotometric . 16 (~~~~=4000 mM-’ err-‘). measurements were pere&man 5240 or a Specord ISS) instrument. Absorption specPhe-NSOH at pH 6, 10 and 12, recorded he Beckman apparatus, were resolved into bands by the method of Powell (apparent) of ground state pK, graphically from the spectroration curves. The error of the iaations was estimated from the maximum tion of the results in the measurement series. ues of pc in the excited state were ded from the kinetic measurements described as a ratio of the rate constants (K= or from the FGrster cycle [14]. rofluorometric measurements were performed using a Perkin-Elmer model 204
59
apparatus supplied with a Perkin-Elmer 56 recorder. Spectrofluorometric titrations were performed as described elsewhere [12]. pH values were adjusted by addition of HCl or NaOH. At low pH (O-l) some quenching of NSOH fluorescence was apparent. True fluorescence intensities (F) were calculated by taking into account the isoemissive points visible in the fluorescence spectra of NSOH conjugates titrated at lower acid concentration. Thus eqn. (2) was applied F0 = F&,(FCI, isalFiscJ
(2)
intensity at a given where Fobs is the fluorescence (low) pH value observed at a given wavelength, Fo,im is the fluorescence intensity at neutral pH (no quenching by Cl- or H+ ions) at the isoemissive wavelength, i.e. the wavelength at which the emission intensity of the ROH form is equa1 to that of the RO-* form (this wavelength is determined as the point at which the fluorescence spectra of a given NSOH derivative taken at various pH values intersect) and Fi, is fluorescence intensity at a given (low) pH value at the isoemissive wavelength. Excited state proton transfer rate constants were obtained from the spectrofluorometric titration results by the method of Weller [14]. Equations (3) and (4) were used (see also eqn. (1))
(4) Thus & was obtained from eqn. (3) where T,, is the lifetime of the first singlet excited state of the ROH* form (see eqn. (l)), & is the fluorescence quantum yield of the ROH form at neutral pH (see Fig_ 2, Section 3) determined by the relative method [18] (see below) and &, and & are the fluorescence quantum yields of pure ROH* and RO-* forms determined by the relative method [18] using, as standard, Schaefer’s acid characterized by emission band positions very similar to those _pf our samples, with &=0.24 and &=0.49 [12]. kpt was obtained by fitting the experimental results to eqn. (4) as a linear dependence of (+/ &,)l,(@l&) on F[H+]. The slope of this pl_ot s =k,,~&,~~,,, with Q and $, measured and k,, determined from eqn. (3), gives the value of kpt. In eqn. (4) ~b is the lifetime of the RO-* form (see eqn. (1)) and F=z~z~(~.O~JO-~)/(~+~~‘) where J is the ionic strength, z1 and z, are the charges of the reactants and 4 and 4’ are the fluorescence quantum yields at a given pH value
60
A. Jankowski
et al. / Proton transfer
of the ROH* and RO-* fluorophore forms respectively. As opposed to thevalue of 4, occurring in eqn. (3) the values 4 and $’ from eqn. (4) should be measured at acidity values sufficiently high to obtain significant changes in the fluorescence spectra with pH (see Fig. 2, Section 3). The lifetime measurements of the first singlet excited state in the compounds studied were performed using an impulse fluorometer LIF 200 (GDR) with a laser source IGT 50 (GDR) (N, u,,,=337 nm). The fluorescence (4 decays were fitted to a single, double or triple exponential (5) The rate constants of dynamic fluorescence quenching (k,) by acrylamide for NSOH groups bound to BSA or Phe were determined from the linear Stern-Volmer plots. The fluorophores bound to proteins may be buried inside the hydrophobic core of the macromolecule and thus may, in some part, be inaccessible to the solvent within the lifetime of the excited state. In this case no linear Stern-Volmer dependence of the fluorescence intensity on the concentration of the ionic quencher is obtained. A modified Stern-Volmer equation enables the fraction of fluorophores accessible to the quencher (fr) to be determined [19]. This value is a measure of the accessibility of the fluorophores to the polar solvent and was employed to charactcrize the position of the sites of binding of NSOH groups in macromolecules using the method of Lehrer [19].
3. Results PAP preparations contain isoenzymes [ZO]. In the chromatographic elution profile of the commercial PAP used in this study three peaks were visible. Only the first, well-resolved fraction of the lowest elution volume was used for modification with JAE-NSOH. The sample of labelled PAP (PAP-NSOH) obtained was homogeneous in polyacrylamide gel electrophoresis. PAP contains one free SH group (cysteine (Cys) 25) situated in the active site [21]. The amount of SH groups in purified PAP decreased after labelling (Table 1) by a quantity corresponding to the amount of bound marker, which was determined independently by a spectrophotometric method. It was concluded that in PAP-NSOH the enzyme was labelled selectively at the SH group
in 2-naphthols
bound
to proteins
positioned in the catalytic niche. However, it should be noted that a small quantity (15%) of PAP remained unlabelled (see Table 1). In the case of BSA-NSOH(S) the protein was labelled using NOAcS-Succ which is expected, in analogy with carbonyl succinimide esters [22, 231, to be more selective than the NOAcSQ used previously [12] towards the amino groups of the protein. It should be noted, however, that our results suggest a possible formation of unstable side products in the reaction of NOAcS-Succ with proteins. The substitution by the fluorescent marker was 0.85 group per protein molecule in PAP-NSOH and 0.7 group per protein molecule in BSANSOH(S) (Table 1). The fluorescence of tryptophan in PAP-NSOH (excited at 280 nm) was identical with that of the unmodified enzyme, suggesting that the native structure of the protein was not disturbed. The same holds for other modified proteins mentioned in this work with the exception of BSA-NSOH(S) where tryptophyl emission is markedly decreased compared with that of the native protein. The fraction of fluorophores accessible to the ionic quencher (JJ) in labelled proteins is given in Table 2. This value is a measure of the exposure of the fluorophoric groups to the solvent. It is evident that in BSA-NSOH(S) the fluorophores are bound preferentially on the surface of the protein molecule and in PAP-NSOH they are located in the interior. The fluorescence spectra and fluorometric titration curves of PAP-NSOH and BSA-NSOH(S) are presented in Figs. 1 and 2 respectively. In Fig. 1 the higher energy band (h,,,.,=363 nm) is due to the ROH* fluorophore form and the lower energy band (A,,,,,= 445 nm) to the RO-* form. The titration curve for BSA-NSOH(S) in analogy with those of other labelled proteins studied previously [12], shows no marked changes in the ROH” and RO-* fluorescence bands at pH 3-7 and both emission bands are observed at pH 1-12. For PAPNSOH, at pH 6-6.5 there is a very rapid rise of the RO-* form emission intensity and at pH 7 only the long-wavelength fluorescence band is observed experimentally. This behaviour is probably caused by deprotonation of the histidine (His) residue positioned close to the active site [24]. Negative logarithms of the equilibrium constants of the proton transfer reaction in the phenolic group (eqn. (1)) of the investigated compounds in the ground (pK,) and excited (pK,*) states are given in Table 3. The ground state pK, values are similar within experimental error ( f 0.2) for most
A. Jankowski et al. I Proton transfer in 2-naphfhok bound to proteins TABLE
1. The amount
of free SH
61
groups and bound marker per protein molecule in PAP and PAP-NSOH
Compound
SH” (M dcm-3,
PAPb (M dcm+)
[NSW (M dcm-‘)
PAP (unlabelled) PAP-NSOH
1.32 x lo-’ 2.96 x10-s
1.37x 1o-5 1.93x 1o-5
0 1.79x10-5
[NSOH]/[PAP]
0.96 0.15
0 0.93
*Q~= 13 600 mM_’ cm-’ was assumed. ‘From gravimetric data. determined from c3353 =4000 r&i-’ cm-l. dJ&perimental error, 5%.
TABLE 2. Accessibility to ionic quencher VI) and fluorescence quantum yields (&, &,) and lifetimes (TV 4$ for NSOH groups bound to proteins and for Phe-NSOH (unprimed symbols for ROH* form and primed symbols for RO-* form) Compound
fi
4
&I
% (ns)
7; (us)
PAP-NSOH BSA-NSOH(S) BSA-NSOHQ) Phe-NSOH
0.32 0.74 0.30
0.048 0.114 0.03 0.25
0.102 0.17 0.13 0.5
3.41 5.2 0.84 7.17
6.73 1155 2.9 10.23
Fig. 2. Spectrofluorometric titration and BSA-NSOH (broken line).
Fig. 1. Fluorescence spectra at pH 6: (1) PAP-NSOH (excitation at 320 nm; concentration, 3 X 10e6 M); (2) BSA-NSOH(S) (excitation at 320 nm; concentration, 3 x lo-” M).
cases. A higher value is obtained only for NSOH groups bound to sodium dodecylsulphate (SDS) micelles where the negative charge density of the micelles explains the effect observed. The excited state pK,” values of the NSOH derivatives differ significantly depending on whether position 6 of the naphthalene ring is substituted by the sulphonamide group. This may be caused by a contribution from the structures with a negative charge density localized at position 6 of the naphthalene ring [27]. Such structures probably contribute more to the excited state than to the ground state wavefunction because of the
of PAP-NSOH
(full line)
greater extent of negative charge migration from the phenolic oxygen to the ring in the excited state. The localization of electrons in position 6 of the naphthalene ring must be hindered for derivatives substituted in this position by a group bearing a negative charge. The opposite effect is expected for compounds substituted at position 6 by an electron-withdrawing group. This may explain the larger decrease in the pK,* values in the 2naphthol derivatives substituted in position 6 by sulphonamide, an electron-withdrawing group, than in analogues bearing a negative charge (Schaefer’s acid). The similar pK,* values for proteinNSOH conjugates and low-molecular-weight NSOH-sulphonamides (Table 3) confirm the view that the NSOH groups in our conjugates are bound to proteins by means of the sulphonamide bond. The interpretation presented above is in agreement with the effects of deprotonation of the sulphonamide nitrogen atom at pH 10-12 on the absorption and fluorescence spectra of Phe-NSOH and other investigated compounds (Table 4). The_ rate con_stants of excited state proton transfer (k,, and kpt, see eqn. (1)) for the NSOH-
A. lankowski
62
et al. I Proton
transferin
2-naphthols bound to proteins
TABLE 3. Negative logarithms of the equilibrium constants of the prcton transfer reactions (eqn. (1)) in the ground excited (pK:) states and rate constants for the excited state reaction (kpt and kpt, see eqn. (1)) Compound
5, (s-’
PK e
(PK.) and
PJ? (Farster
M-’ 1”x lo+?)
cycle) ‘2.Naphthol” Schaefer’s acidb Phe-NSOH NH,-NSOH EACA-NSOH Gly-NSOH SDS-NSOH PAP-NSOH BSA-NSOH(S) BSA-NSOH(1) ‘Data from ref. 25. bData from ref. 26. ‘Experimental error,
9.46 9.1 9.2 8.9 9.2 9.1 11.05 8.7 9.3 9.7
a.72 8.5 7.92 9.98 0.27 8.17 1.44 0.82
Absorption
Solution
Hz0 (PH 6) 0.1 n KOH (pH 10) 1 n KOH (pH 13) investigated
2.5 1.6 0.6 0.7
0.6
* 0.2.
TABLE 4. Effect of ionization of the sulphonamide of Phe-NSOH”, resolved into gaussian components,
“Other
2.8 1.66 0.51 0.7 0.48 0.49 3.90 0.48 1.14 0.32
694 406 39.6 56.1 33.6 43.3 3900 72.8 38.5 18.7
1.1 10.2 12.1 * 1 11.2*2 11.1*1 13.9*1 0.4*1 23.9+2 2.7 * 1 10.4
sulphonamides
nitrogen atom on the positions of the band maxima in the absorption and in the fluorescence spectrum spectrum
Fluorescence spectrum (RO-*)
maxima
(nm)
‘I-% (nm)
$n)
$)
298.4 344.1 348.4
279.6 305.7 301.1
236.4 246.0 246.2
show similar effects but the spectra
conj_ugates studied are given in Table 3. The error of k,, determination (given in Table 3) was estimated from the maximum deviation in the fluorometric measurements (intensity-and lifetime}. The error of determination of k,, was higher because of the necessary addition of HCl (see Section 2.5). This error has not been estimated. For the low-molecular-weight sulphona_mides studied, the deprotonation rate constants k,, are practically identical within experimental error. For the NSOH groups bound to the surface of the protein in BSA-NSOH(S), the kpl value is much lower and, for NSOH groups in the catalytic site of PAP-NSOH, it is higher than for the lowmolecular-weight analogues. In protein-NSOH conjugates the fluorophoric groups are shielded, to a large extent, from the solvent by neighbouring groups of the macromolecule. This implies a decrease in the steric accessibility for the proton transfer reaction to the solvent for the groups bound to a protein. If this reaction were diffusion controlled, a higher reaction rate would be expected for sites of better accessibility to the solvent, such as those in BSA-
spectrum
were not resolved
into separate
445 443 436 components.
NSOH(S), than for PAP-NSOH where the accessibility of the fluorophore is much lower (Table 2). The present results suggest that, for fluorophores buried inside a protein interior, e.g. in PAPNSOH, proton transfer through preformed hydrogen bonds takes place. To verify the dependence of the proton transfer rate on the accessibility of the proton donor to the solvent, we studied the dependence of the excited state proton transfer in NSOH-protein conjugates on the rate of water diffusion by another method. An increase in protein concentration above a certain limit causes aggregation of the macromolecules. This will lead to a decrease in the probability of encounter of water molecules with the excited fluorophores. If this effect influences the rate of proton transfer, an increasing protein concentration should cause definite changes in the fluorescence spectra. To test the influence of water diffusion on the proton transfer reaction, we investigated the effect of an addition of unlabelled protein on the fluorescence of prokin-NSOII conjugates (Fig. 3). In this experiment the influence of increasing sample turbidity on the
A. Jankowski e[ al. I Proton
transferin 2-naphtholr bound to proteins
63
centration is nearly linear, as for low-molecularweight sulphonamides. Therefore a Stern-Vohner plot can be used in both cases. To compare the exposure of fluorophores in BSA-NSOH(S) with those in low-molecular-weight NSOH-sulphonamides we used the fluorescence quenching wnstants by acrylamide (k,) given in Table 5 k, = K&o 02
_
2 5 ,
100
x I‘
I
I
. ‘200
!
!
s
PROTEIN Fig. 3. Dependence of Q at the peak maxima tonated (RO-*) forms (1) BSA-NSOH(S); (2)
1.
’ ‘330
:I
I
a
::
Fwmll
,
CONCENTRATION
the ratio of the fluorescence intensities of the protonated (ROH*) and deproan the addition of unlabelled protein: PAP-NSOH.
fluorescence spectra was eliminated by subtracting from the spectra the baseline obtained by inserting in the exciting light beam a solution of unlabelled BSA of equal concentration as the sample investigated. The unlabelled BSA shows no absorbance at the exciting wavelength (320 nm). It can be seen from Fig. 3 that PAP-NSOH fluorescence is insensitive to a rise in protein concentration. However, BSA-NSOH(S) shows a marked enhancement of the fluorescence intensity (F) of the protonated form (ROH*) and a decrease in emission from the deprotonated form (RO-*) with increasing protein concentration. This is illustrated in Fig. 3 by an increase in the ratio F ROHsIFRO-*with increasing protein concentration. We conclude that the excited state proton transfer reaction in BSA-NSOH(S), but not in PAP-NSOH, is sensitive to the rate of diffusion of water towards the excited fluorophores. Thus a decrease in the diffusion coefficient for water causes a decrease in the rate of excited state proton transfer in this sample. Thus a reduction in the rate of excited state proton transfer in BSA-NSOH(S) compared with the value for low-molecular-weight sulphonamides (Table 3) must be at least partly due to the decrease in the accessibility of the Uuorophores bound to the protein surface. A comparison of the exposure of the fluorophores bound to BSA with those in lowermolecular-weight sulphonamides cannot be performed by the method of Lehrer [19] (see Section 2.5). However, because the fraction of fluorophores accessible to the ionic quencher (fi) in BSANSOH(S) is high (Table 2), the dependence of the fluorescence intensity on the quencher con-
(6)
where I&, is the Stem-Vohner constant and Q is the lifetime of the excited state of the pure ROH* form. From the results in Table 5 we conclude that the 50% decrease in the accessibility of NSOH groups in BSA-NSOH(S) with respect to the value for Phe-NSOH (and other low-molecular-weight analogues) canno_t fully account for the 70% reduction in the kpf value (Table 3). The determination of the reason for the reduction in the proton transfer rate for BSA-NSOH(S) requires further investigation. It was of inte_rest to determine whether the dependence of k,, on pKf for the investigated compounds (Table 3) would yield a similar fiicture to that of many other reactions of organic (acids, including the excited state data for 2-naphthol analogues [6, 281. The relationship between log k,, and log K,* for the compounds studied (Fig. 4) shows that the data for low-molecular-weight NSOH-sulphonamides can be fitted to the curve for other 2-naphthol derivatives of low molecular weight. In this way a plot very similar to that published previously [28, 291 is obtained. This suggests that the excited state proton transfer in low-molecular-weight analogues follows the same mechanism, and is characterized by parameters such as the proton vibrational strength in the initial and final states of the reaction, the energy of medium reorganization, the proton tunnelling probability, etc. [29, 301. The data for the majority of macromoleculeNSOH conjugates (NSOH-protein and NSOHSDS) cannot be fitted to the plot in Fig. 4. Only the result for PAP-NSOH (11, Fig. 4) fits the curve constructed for low-molecular-weight analogues of 2-naphthol. This is unexpected because TABLE 5. Rate constants (k.J by acrylamide
of dynamic
fluorescence
quenching
Compound
J&v
70 w
kqx lo-’
Phe-NSOH BSA-NSOH(S)
2.00 0.745
7.17 5.20
2.78 1.43
Symbols are explained
in the text.
64
A. lankowski
et al. I Proton
transferin 2-naphth&
Fig. 4. Dependence of log &, on log K: (Breinstedrelation): (1) 2-naphthol; (2) 1-naphthol; (3) 2-naphthol-6.sulphonate; (4) PheNSOH; (5) Gly-NSOH; (6) SDS-NSOH, (7) 7-methyl-2.naphthol; (8) 6.methyl-2.naphthob (9) 1.chloro-Z-naphthol; (10) l-bromo2-naphthol; (11) PAP-NSOH; (12) BSA-NSOH(I); (13) BSANSOH(S); (14) BSA-NSOH(A). Filled circles, low-molecularweight derivatives of Z-naphthol; open circles, macromoleculeNSOH conjugates. Data for samples t-3 were obtained from refs. 25 and 26, for samples 4-6 and 11-14 from our results and for samples 7-10 from ref. 28. Samples 12 and 14 are described in detail elsewhere [12].
of the position of the NSOH group in the catalytic site in PAP-NSOH. This problem requires further study. From the data in Fig. 4 it can be concluded that the majority of macromolecule-NSOH conjugates are characterized by a different mechanism of excited state proton transfer to the low-molecular-weight analogues.
4. Discussion In this work we studied the rates of excited state proton transfer in fluorophores bound selectively in the active site of PAP in PAP-NSOH. For the other sample studied (BSA-NSOH(S)) the position of the proton donor was less precisely known, but it was characterized in terms of the exposure of the fluorescent groups to the solvent. Some results for other NSOH-protein conjugates obtained previously [12] were used for comparison. The high rate of proton transfer for groups of low accessibility to water (Table 3) is most probably due to the mechanism of proton transfer through intramolecular hydrogen bonds. Chains of hydrogen bonds may be formed by side groups of some amino acids or by the peptide backbone [31, 321. There is also a possibility of proton transfer through chains of hydrogen-bonded water molecules inside the protein macromolecule [24, 331. For proton transfer through chains of hydrogen bonds, which are more stable than those in bulk water, a very
bound to proteins
high rate and a transmission coefficient close to unity may be expected [32], Two ionogenic groups (His 159 and aspartic acid (Asp) 175), which can act as proton acceptors, are present in the active site of PAP. In unmodified PAP, Asp 175 and His 159 are probably engaged in hydrogen bonding [21]. This statement, based on crystallographic data [24], in connection with the low ground state pK, value of PAP-NSOH (Table 3), indicates that the NSOH group bound in the active site of PAP is not engaged in intramolecular hydrogen bonding to Asp 175 or His 159. The Asp 148 residue is separated by too large a distance to allow hydrogen bonding with the NSOH group bound to Cys 25. Therefore, in the case of PAP-NSOH, proton transport through internal hydrogen-bonded water molecules of the protein is more probable than transfer through the intramolecular hydrogen bonds of the protein. Proton transfer through chains of four hydrogenbonded water molecules in bulk water is also responsible for the excited state deprotonation of low-molecular-weight 2.naphthol analogues in water solution [34]. The reason for the enhancement of the rate of proton transfer for NSOH groups buried inside the protein core relative to that for low-molecular-weight analogues may be the reduction in the energy of medium reorganization (E,) [29, 30, 33-361 and/or special ordering of internal water molecules surrounded by the protein network 133, 37, 381. The results of the fluorometric titration (Fig. 2) suggest a role for the partial electrostatic charge of His 159, which is expected to release a proton at pH 6 [24, 331, in increasing the rate of proton transfer in PAPNSOH. A comparison of the &, values for PAP-NSOH and BSA-NSOH(1) (a sample described in detail elsewhere [12]) with the data on solvent accessibility (Table 2) shows that the degree of enhancement of the proton transfer rate for fluorophores situated in the hydrophobic core of proteins is different for groups of comparable solvent accessibility. We suppose that, in BSANSOH(1) (see ref. 12), where the labelling was non-homogeneous but the NSOH groups were preferentially located inside the hydrophobic core, proton transfer through the intramolecular hydrogen bonds of the protein was significant. In contrast, the k,, values for BSA-NSOH(S) and other conjugates characterized by highly accessible NSOH groups are similar (see Tables 2 and 3 and table 2, ref. 12). For NSOH groups bound on the surface of macromolecules some immobilization of water at the interface boundary
A. Jankowski et al. I Pmton transfer in 2-naphtholrr bound to proteins
may influence proton transfer [lo, 331. Because of the non-zero surface charge of the protein molecule the probability of geminal recombination of the proton immediately after its release from the excited NSOH group must be taken into account [lo]. Both of these effects (immobilization of water and geminal recombination) may be responsible for the decrease in the proton transfer rate for NSOH groups bound to the surface of proteins compared with the value for the lowmolecular-weight analogues. We were unable to estimate experimentally the contribution of geminal recombination in the fluorescence spectra of our samples, but it can be supposed that its role is not very significant since the pI(,* values determined from the Flirster cycle are roughly coincident with the data obtained from our kinetic measurements (Table 3). Gutman et al. [7, lo] investigated proton transfer reactions in chromophores bound to macromolecular systems such as proteins, micelles and membranes. These workers obtained, for proteinbound flyorophores, higher protonation rate constants (k,f), and comparable deprotonation rate constants (k,,) with our results for surface-bound NSOH groups. The position of the proton donor and acceptor in terms of the accessibility to the solvent was not specified in the work of Gutman et al. [7, IO].
biological phenomena, further experimental evidence on the mechanism of proton transfer in biological systems is required. Acknowledgments This work was supported by a grant from the Committee of Scientific Research (Z-0760-91-01) and by the Technical University of Wroclaw. References 1 P. Bayer, B. Chance,
2 3 4 5 6 7 8 9 10 11
12
5. Conclusions The following conclusions may be drawn from our results. (1) The rate of proton transfer for groups located in the hydrophobic core of proteins is enhanced relative to that for low-molecular-weight analogues. This conclusion confirms a similar opinion reached previously 111-131. (2) The enhancement of the reaction rate for internally bound NSOH groups is probably due to proton transfer via the intramolecular hydrogen bonds of the protein or via chains of internal, structural water molecules. (3) A decrease in the rate of proton transfer for surface-bound groups compared with low-molecular-weight analogues is due mainly, but not exclusively, to a reduction in the accessibility to water. It has been postulated on theoretical grounds [36] that the enhancement of the rate of proton transfer inside protein molecules is of major importance in enzymatic Catalysis. As proton transfer reactions also play an important role in other
6.5
13 14 15 16 17 18 19 20 21 22 23 24 2.5 26 27 28
29
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