Flavin mononucleotide fluorescence intensity decay in concentrated aqueous solutions

Chemical Physics Letters 439 (2007) 151–156 www.elsevier.com/locate/cplett

Flavin mononucleotide fluorescence intensity decay in concentrated aqueous solutions Hanna Grajek a, Ignacy Gryczynski b, Piotr Bojarski c,*, Zygmunt Gryczynski b, Shashank Bharill b, Leszek Kułak d a Department of Physics and Biophysics, University of Warmia and Masury in Olsztyn, Poland Department of Cell Biology and Genetics, Health Science Center, University of North Texas, USA c University of Gdan´sk, Institute of Experimental Physics, Wita Stwosza 57, 80 952 Gdan´sk, Poland Technical University of Gdan´sk, Department of Technical Physics and Applied Mathematics, Narutowicza 11/12, 80-952, Gdan´sk, Poland b

d

Received 22 February 2007 Available online 20 March 2007

Abstract Time-resolved fluorescence and steady-state absorption measurements of FMN aqueous solutions were carried out over very broad dye concentration range. Efficient formation of FMN dimers with simultaneous absence of higher order aggregates even at highest concentrations was found from absorption spectra measurements. It was found that fluorescence intensity decays are strongly accelerated in the presence of dimers due to excitation energy trapping and become nonexponential. Mean localization time of excitation energy and mean number of its jumps between FMN molecules were calculated as a function of concentration.  2007 Elsevier B.V. All rights reserved.

1. Introduction Flavins are very important biological molecules. As coenzymes and photoreceptors they play an important role in numerous biological processes [1–3]. The illumination by blue and near ultraviolet light, absorbed by flavins, leads to several processes in plants such as chloroplast migration, the opening of the stomatal guard cells, phototropism and others. This is why their properties are widely studied with the spectroscopic techniques. Recently, a lot of work has been devoted to photocycle studies of light, oxygen and voltage sensitive (LOV) domains in which flavins participate [4–8]. The mechanisms of these reactions and the interactions between various LOV domains are still far from full understanding. In particular, it is not clear whether monomers or dimers of flavins are responsible for photoreception. However, the results presented in [3,9] suggest that these may be the *

Corresponding author. Fax: +48 58 341 31 75. E-mail address: fi[email protected] (P. Bojarski).

0009-2614/$ - see front matter  2007 Elsevier B.V. All rights reserved. doi:10.1016/j.cplett.2007.03.042

dimers since the action spectra of chloroplasts and other organisms resemble more dimer than monomer absorption spectra. Moreover, the existence of FMN dimers in biological [10] and photoreceptor systems [11] has been shown. From the physical point of view flavin mononucleotide (FMN) solutions form a very interesting and exceptional model system for study of energy transfer and aggregation processes since FMN practically does not form aggregates of higher order than dimers [12,9,13]. FMN dimers in rigid solution have also a unique feature that they are fluorescent and form imperfect traps for excitation energy transferred from monomers [14]. Therefore, such a system can be considered as strictly two-component and in the case of rigid solutions described nicely by an analytical model of forward and reverse multistep energy transfer and/or respective Monte-Carlo simulation [15]. However, this fluorescence seems to disappear in aqueous solutions [16,17]. Simultaneously, material diffusion can significantly affect energy transport mechanism resulting in inadequacy of the model mentioned [16].

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The FMN aggregation studies performed so far in aqueous solutions based mostly on steady-state absorption and steady-state concentration quenching measurements. In these works the structure of dimers and some FMN spectroscopic properties have been determined [12,13,16–18]. As these techniques are not as sensitive as time-resolved ones they cannot provide detailed information on multistep energy transfer and trapping by dimers. Until now no systematic study of dimer formation on FMN fluorescence intensity decay has been performed. The task of this Letter is to fill partly this gap and provide information on the mechanism of monomer–dimer energy transfer for FMN in water using both time-resolved and steady-state techniques. 2. Materials and methods Flavin mononucleotide (riboflavin-5 0 -monophosphate, sodium salt; C17H20N4NaO9P Æ 2H2O), analytically pure from Fluka AG was used without further purification. The respective FMN concentrations were prepared in deionized water (66 mM potassium phosphate buffer, pH 7.0) by using the weighing dilution method with an accuracy of 0.1% over the concentration range from 1.05 · 105 M to 2.84 · 101 M. The absorption spectra were measured using the Beckman Model DU520, Varian/Cary 50 spectrophotometer. Mean fluorescence lifetimes were determined from fluorescence intensity decays using the Fluotime 200 spectrofluorometer (Picoquant). In the experiment violet (k = 473 nm), pulsed picosecond semiconductor laser: LDH-C 470 head (PicoQuant GmbH, Germany, FWHM of approximately 70 ps) was used as a light source. Fluorescence light was collected by the ultrafast microchannel plate photomultiplier (H5783P Hamamatsu Photonics K.K., Japan). The analysis of fluorescence intensity decays was carried out using Fluofit software (Picoquant). Multiexponential fluorescence decay model was employed. The fit quality was judged by the v2 value and the distribution of residuals: n 1 X 2 wk ½gðtk Þ  gteor ðtk Þ ð1Þ v2 ¼ n  p k¼1 pffiffiffiffiffi Rw ðtk Þ ¼ wk rk ð2Þ rk ¼ gðtk Þ  gteor ðtk Þ;

ð3Þ

where p is the number of the fitted parameters, n is the number of experimental points, g(tk) and gtheor(tk) is the value of experimentally measured and theoretical fluorescence intensity, respectively, and wk = [g(tk)]1 is the weighting factor. 3. Monte-Carlo simulations To obtain theoretical values of mean fluorescence lifetime, the mean localization time of excitation energy on FMN monomer molecules and the mean number of energy

jumps between FMN molecules Monte-Carlo simulations were carried out. Since detailed procedure of simulation has been reported previously [19], here only a brief summary is given. In a simulation, NM monomers of concentration CM and ND dimers of concentration CD, are randomly distributed in a three-dimensional cube. The effect of the finite size of the system is minimized by periodic boundary conditions with minimum image convention. The concentration courses of mean fluorescence lifetime, mean localization time and the number of energy jumps are obtained by rescaling critical radii for energy transfer and keeping the length of the cube edge equal to 1. The pseudo-random number generator, which passed several statistical tests was also verified by checking the simulated statistical clusters concentration against the analytically expected value. The simulated configurations were sampled until the relative variance of the quantities mentioned attained less than 0.1%. In our case we used the ‘step by step’ algorithm. It consists in use of the random number generator for the cyclic formulation of answers to two questions: when any of the assumed luminescent processes takes place and what kind of process it is. The simulation algorithm includes the following main steps: 1 The coordinates of a primarily excited molecule are determined. This molecule can be deactivated through: (P1) process 1: M* ! M photon emission or nonradiative energy transition, with the rate 1/s0M. (P2) process 2: M* + M ! M + M*, energy migration (energy transfer to the molecules of the same kind), with the transfer rate wMM ji . (P3) process 3: M* + D ! M + D*, nonradiative energy transfer from the excited monomer to a dimer, with the transfer rate wMD ji . 2 If ith monomer molecule is excited, the following total transfer rates are calculated: c1i ¼ 1=s0M ;

c2i ¼

NM X

wMM ji ;

c3i ¼

NM þN D X

wMD ji :

ð4Þ

j¼N M þ1

j¼1;i6¼j

Otherwise, when ith dimer is excited, the values of c01i ¼ 1=s0D ;

c02i ¼

NM þN D X

wDD ji

ð5Þ

j¼N M þ1;i6¼j

are calculated. 3 The time, at which any of the investigated processes occurs (cp. step 1), is calculated by inverting the distribution function of the probability pi(t, Pk)dt that if at time t the ith molecule is excited, then the process Pk takes place over the time interval (t, t + dt): pi ðtÞ ¼

3 X k¼1

pðt; P k Þ ¼ ci expðci tÞ;

ð6Þ

H. Grajek et al. / Chemical Physics Letters 439 (2007) 151–156

153

where ci ¼ c1i þ c2i þ c3i

ð7Þ

For this purpose the random number, r1i, is generated and the time at which any process takes place is obtained by inverting the distribution function of probability pi(t, Pk) Z ti pi ðtÞdt ¼ r1i ; i:e: ti ¼ ð1=ai Þ ln r1i ð8Þ 0

4 In this step, it is determined which process took place at the time ti. By generating the next random number, r2i, such a value of index k can be found for which the following inequality is satisfied: k1 X j¼1

cji < r2i ci 6

k X

k ¼ 1; 2; 3

cji ;

ð9Þ

j¼1

If k = 1, then the activated molecule is deactivated by a photon emission or nonradiative transition which means that this pass of simulation is finished. If k = 2 or k = 3, the energy migration or transfer process takes place, and it is necessary to determine currently excited molecule. For this reason, the third random number, r3i, is generated and the value of index n is found which fulfills the following inequality: n1 X j¼1

wMM < r3i c2i 6 ji

n X

wMM ji ;

for n 6 N M ;

ð10Þ

j¼1

where n is the number of next activated monomer or dimer molecule. Then, after inserting the value of n for the index i, the simulation goes to step 2. The simulation run is finished when the process with k = 1 occurs in step 4. After that a new simulation for a new monomer and dimer spatial configuration runs. 4. Results and discussion Fig. 1 shows the absorption spectra of FMN in aqueous solutions measured over the concentration range from 1.05 · 105 M to 2.84 · 101 M at room temperature 298.2 K. This concentration range is significantly wider than previously used (C = 1.8 · 102 M, [12]) resulting in much more pronounced concentration changes in absorption spectra. Previously [12,18] the maximal extinction coefficients e (of the absorption band I) – changed from 11,800 to 10,000 M1 cm1 and were visibly different from the calculated pure FMN dimer absorption spectrum for which eD  8000 M1 cm1. This time, the maximal extinction coefficient strongly decreases with concentration increase down to e = 8400 (±200) M1 cm1. It can be seen that the shape of absorption spectra is different for C P 8.05 · 102 M and resembles closely dimer absorption spectrum of FMN. The dimerization constant K, the pure absorption spectra of the FMN monomer M(k) and dimer D(k) have been determined in [12,18], based on the Fo¨rster–Levshin modified method. For clarity, Fig. 2 shows the FMN monomer spectrum, determined in this work

Fig. 1. Concentration changes of FMN absorption in aqueous solution (pH = 7.0). I and II – absorption bands corresponding to S1 S0 and S2 S0, respectively.

which occurred identical with the FMN spectrum measured at low concentration as well as the spectrum of the most concentrated sample at C = 2.84 · 101 M, which is very close to the pure spectrum of FMN dimer D(k). Table 1 shows the values of monomer and dimer concentrations as well as the monomer fractions (X) and the fraction of molecules appearing in the dimer form (1  X) at subsequent FMN concentrations for all samples. These values were determined using the following relations: pffiffiffiffiffiffiffiffiffiffi 1 CM = XÆC, CD = [(1  X)/2]ÆC, where X ¼ 1þ8KC is the 4KC monomer fraction and K is dimerization constant. The calculations were performed for K = 118 M1 (at T = 298.2 K [12,18]). From Table 1 it can be seen that for C = 1.98 102 M, CD  CM, whereas for the highest concentration C = 2.84 · 101 M around 90% of all molecules in the solution is dimerized. For this latter case the absorption spectra of the most concentrated samples are practically identical to that of the pure dimer: (1) the location of band maximum connected with the transition S2 S0 is shifted towards shorter wavelengths (Table 2), as it is in the case of the dimer spectrum, (2) the long wave S1 band exhibits two transitions corresponding to H (near 446 nm) and J

Fig. 2. FMN monomer and dimer absorption spectra as well as total absorption spectrum of the most concentrated FMN sample.

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H. Grajek et al. / Chemical Physics Letters 439 (2007) 151–156

Table 1 Total concentration C, monomer CM and dimer concentration CD, respectively as well as monomer X and dimer 1  X fraction, respectively C (M)

X

1X

CM (M)

CD (M)

1.05 · 105 1.00 · 104 1.00 · 103 2.36 · 103 9.94 · 103 1.98 · 102 3.35 · 102 5.92 · 102 8.05 · 102 1.05 · 101 1.34 · 101 1.96 · 101 2.84 · 101

0.998 0.977 0.835 0.715 0.474 0.368 0.298 0.234 0.205 0.182 0.163 0.137 0.115

0.002 0.025 0.165 0.285 0.526 0.632 0.702 0.766 0.795 0.818 0.837 0.863 0.885

1.05 · 105 9.77 · 105 8.35 · 104 1.69 · 103 4.71 · 103 7.28 · 103 9.98 · 103 1.39 · 102 1.65 · 102 1.91 · 102 2.18 · 102 2.68 · 102 3.26 · 102

1.29 · 108 1.13 · 106 8.23 · 106 3.36 · 104 2.62 · 103 6.26 · 103 1.18 · 102 2.27 · 102 3.02 · 102 4.30 · 102 5.61 · 102 8.46 · 102 1.26 · 101

Table 2 The location of absorption band maxima for FMN in H2O

ratio for absorption bands (I and II)

distance between the molecules

water molecules so that there is no possibility to bind another one FMN monomer. Fig. 3 shows the fluorescence intensity decays for a couple of FMN concentrations ranging from 1.05 · 105 M to 2.84 · 101 M. It can be seen from the figure that at low concentrations, in the absence of energy migration and trapping, the FMN fluorescence intensity decay is single exponential. With the increase in concentration, starting from C = 1.98 · 102 M the decay is accelerated with initially slight deviations from single exponential character. These deviations are much more evident at highest concentrations and the course of the decay curve is getting more complex. The acceleration of the fluorescence intensity decay and its nonexponential character results from intensive trapping by dimers preceded by energy migration [21] as well as by the contribution of material diffusion. Since there is no analytical model accounting for multistep energy migration, trapping and material diffusion at the same time, we decided for now to describe formally the results obtained within the multiexponential model. The decay curves for concentrations between 1 · 103 M and 102 M have been fitted to two – exponential function, however, satisfactory fit for concentrations equal or higher than 1.98 · 102 M could be only obtained in terms of three exponential model, which reflects increasing complexity of the fluorescence decay function with growing concentration. Based on absorption and fluorescence spectra measurements we found that critical distance for energy migration M* ! M and transfer M* ! D for FMN in water amounts ˚ and R0MD = 21.54 A ˚ , respectively, to R0MM = 20.33 A which corresponds to the following critical concentrations: C0MM = 4.7 · 102 M for energy migration and C0MD = 3.96 · 102 M for energy transfer. These critical distances and corresponding concentrations were obtained from the well known Fo¨rster formula [22,16]:

I/II = 1.09 ± 0.02 I/II = 1.08 ± 0.02 I/II = 1.06 ± 0.02 (I/II)D = 1.088 (I/II)M = 1.21

14.35 11.24 11.17

R60MX ¼

ð11Þ

C 0MX

ð12Þ

Transition

S2 S0 (nm)

S1 S0 (nm)

S2 S0 (nm)

FMN monomer C = 2.84 · 101 M FMN dimer

372 365 365

446 446 (H band) 446 (H band)

480 (J band) 480 (J band)

(near 480 nm) as it is in the dimer band [13]. Simultaneously, it can be easily found that the ratio of maximal extinction coefficients for both absorption bands (I and II) I/II for extremely high concentrations: 1.34 · 101 M, 1.96 · 101 M and 2.84 · 101 M is approximately the same as that for the dimer spectrum (I/II)D, but it deviates from the ratio (I/II)M calculated for the monomer (cp. Table 3). The table shows also the mean distance R between the molecules for these concentrations calculated from: R3 = 3 · 1027/4pNAC, where NA [mol1] ˚ ], denotes the Avogadro constant, R is expressed in [A and C in [M]). These values exceed those of linear dimen˚ ), but are somewhat sions of monomers (r = 9.4–9.6 A ˚ [20]). Such smaller than that of the dimer (r = 21.14 A small values of R confirm that almost all the molecules are dimerized at the highest FMN concentration. On the other hand, we have not observed any additional changes in shape or shift of absorption spectra which means that FMN does not form higher order aggregates. This is consistent with the results reported in [12,13], where it has been proven that stacking FMN dimers are stabilized by four Table 3 Extinction coefficient ratio for both absorption bands and the mean distance between the molecules at several FMN concentrations ˚ ]-the mean C (M) Extinction coefficient R [A

1.34 · 101 1.96 · 101 2.84 · 101 FMN dimer FMN monomer

Fig. 3. FMN fluorescence intensity decays measured for several dye concentrations.

9000ðln 10Þhj2 ig0M I MX 128p5 n4 N ¼ 4:23  1010 n2 ðhj2 ig0M I MX Þ1=2

H. Grajek et al. / Chemical Physics Letters 439 (2007) 151–156

155

where n denotes the refractive index of the medium, N, the Avogadro’s number, g0M, the absolute quantum yield of the donor (monomer) in the absence of any energy transfer processes, Æj2æ, the averaged orientation factor dependent on the mutual molecular alignment, and IMX, the overlap integral of the donor fluorescence spectrum F M ðmÞ (monomer) with the acceptor absorption spectrum of the monomer eM ðmÞ or dimer eD ðmÞ: Z 1 F M ðmÞeX ðmÞm4 dm ð13Þ I MX ¼ 0

whereR the donor emission spectral distribution is normal1 ized: 0 F M ðmÞ dm ¼ 1. From Fig. 3 it follows, however, that the intensity decay is significantly accelerated even at C = 1.98 · 102 M, i.e., at the concentration significantly lower than both calculated critical concentrations. This fact is due to material diffusion, which enhances significantly the probability of energy migration and transfer [27]. Based on the fluorescence intensity decay analysis it is also possible to obtain mean fluorescence lifetimes at different FMN concentrations (cp. Table 4) using the formula: n n n .X X X s¼ ai s2i ai s i ¼ fi si ð14Þ i¼1

i¼1

i¼1

As can be seen from the table the mean fluorescence lifetime of FMN at low concentration s = 4.67 ns, which is in a very good agreement with data obtained by other authors [23–26]. As expected, with the increase in concentration the mean fluorescence lifetime strongly decreases and at the highest concentration it attains 0.43 ns. Fig. 4a shows the comparison between the mean fluorescence lifetime measured (e) and calculated by Monte-Carlo simulation (j) as well as calculated mean localization time (d). The localization time is calculated as sloc = s0/(n + 1), where n is the mean number of excitation energy jumps. Somewhat shorter experimental lifetimes result from the effect of material diffusion, which enhances the efficiency of multistep energy migration. It should be noted here that the mean fluorescence lifetime

Fig. 4. (a) Experimental (e) and simulated (j) mean fluorescence lifetime as well as mean localization time (d) of excitation energy versus FMN concentration. (b) Mean numbers of excitation energy jumps versus FMN concentration: total number (m), between the monomers (s) and monomer to dimer (d).

may be significantly longer than the mean localization time of excitation energy on a monomer molecule. This is because excitation energy may randomly walk within the

Table 4 The parameters of FMN fluorescence decay in aqueous solutions (pH 7.0) fitted to the sum of exponentials and the values of mean FMN fluorescence lifetime (s) obtained from (14) at kex = 470 nm and kem = 530 nm, at T = 293 K C (M) 5

1.05 · 10 1 · 104 1 · 103 2.36 · 103 9.94 · 103 1.98 · 102 3.35 · 102 5.92 · 102 8.05 · 102 1.05 · 102 1.34 · 102 1.96 · 102 2.84 · 102

f1

s1 (ns)

1.0 1.0 0.987 0.984 0.943 0.913 0.841 0.624 0.302 0.487 0.385 0.043 0.370

4.67 4.68 4.66 4.58 4.15 3.56 2.95 2.28 0.96 1.47 1.24 2.31 0.50

f2

0.013 0.016 0.050 0.076 0.143 0.325 0.061 0.417 0.493 0.660 0.139

s2 (ns)

0.05 1.11 1.87 1.60 1.43 1.37 0.21 0.82 0.68 0.62 0.01

f3

0.007 0.011 0.016 0.051 0.637 0.096 0.122 0.297 0.444

s3 (ns)

0.25 0.26 0.23 0.32 1.79 0.24 0.19 0.20 0.19

f4

0.047

s4 (ns)

3.28

v2

s (ns)

1.168 1.057 1.059 1.022 1.120 0.993 1.073 1.039 1.126 1.106 0.991

4.67 4.68 4.59 4.52 4.01 3.38 2.69 1.89 1.44 1.08 0.84 0.57 0.43

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H. Grajek et al. / Chemical Physics Letters 439 (2007) 151–156

set of monomers and be transferred finally to dimers [21] before the act of emission or quenching. Such a difference is reflected also in Fig. 4a, where the concentration dependence of FMN mean fluorescence lifetime accompanied by a similar change in the mean localization time is shown. For systems in which energy migration takes place, the direct measurement of localization time is impossible. This is because mean fluorescence lifetime is not affected by energy migration between identical molecules, but only by its transfer to an unlike molecule. To obtain mean localization time sloc the total number of excitation energy jumps must be determined. This can be made using the Monte-Carlo simulation technique. It can be seen from the Fig. 4a that the mean localization time diminishes strongly with increasing FMN concentration and at high concentrations attains values much shorter than the mean fluorescence lifetime. An interesting result can be seen in Fig. 4b. The number of excitation energy jumps initially increases as concentration grows because the probability of energy migration increases, and the number of dimers (traps) is relatively small. However, at highest concentrations due to strong dimerization, energy migration is weakened due to the effective competition from direct energy transfer to dimers. Therefore, the overall number of excitation energy jumps in the system studied starts to decrease at sufficiently high concentration. At the highest concentration the number of jumps within the monomer set is lower than 1 and we deal with a case close the Fo¨rster one. 5. Conclusions

the effect of material diffusion into quantitative analysis. We are going also to specify conditions under which FMN dimers can be treated as perfect traps. Acknowledgement This Letter has been supported by Grant BW 522-07060206 and BW 5200-5-0294-7. We would like to thank Texas Emerging Technologies Fund for supporting this work. References [1] [2] [3] [4]

[5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15]

Temporal- and concentration-dependent studies of FMN in water exhibit efficient FMN dimer formation. However, even at very high concentrations no higher order aggregates were found. Energy transfer from monomers to dimers is generally preceded by energy migration. The efficiency of this latter process initially increases with concentration, but upon dimer formation it starts to drop as a result of growing competition originating from direct energy transfer from monomers to dimers. The calculations of energy hops number between molecules versus concentration supports this. The comparison between experimentally obtained and simulated values of mean fluorescence lifetimes reveal that the effect of material diffusion enhances the efficiency of energy migration. In the subsequent study, we are going to discuss temperature and viscosity effects on energy transport and include

[16] [17] [18] [19] [20] [21] [22] [23] [24] [25] [26] [27]

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