PIERS ONLINE, VOL. 5, NO. 6, 2009 561 Microwave Effect on Proteins in Solution — Fluorescence Polarization Studies I. Barak, M. Golosovsky, and D. Davidov The Racah Institute of Physics, The Hebrew University of Jerusalem Jerusalem 91904, Israel Abstract— We studied the fluorescence emission of the Enhanced Green Fluorescent Protein (EGFP) in aqueous solution under continuous microwave irradiation with a well-defined field pattern (a TE 011 microwave cavity operating at 9.5GHz). We focused on polarization of the emitted light and measured the spectrum of the fluorescence emission anisotropy. We found that in the spectral range 500–540 nm the microwave-induced effect can be reduced to heating, while in the spectral range 540–560 nm, the microwave-induced effect differs from that resulting from conventional heating. 1. INTRODUCTION Whether or not microwave irradiation can exert a non thermal effect on biomolecules is a con- troversial issue [1,2]. Calculations showed that the resonant excitation of a molecule in aqueous solutions, as well as the thermal gradient between the molecule and its surroundings, is negligibly small at microwave frequencies [3,4], in such a way that the non thermal effect of microwave fields on proteins may be discernible only at extremely high electric field > 10 7 V/m [5, 6]. The empirical evidence for the non thermal microwave effect on biomolecules is inconclusive. The X -ray studies of the microwave effect on the protein conformation in the crystalline form [7]; fluorescence studies of organic fluorophores [8]; the functioning of ion channels in living cells under microwave irradi- ation [9] did not reveal non thermal microwave effects. Several other studies [10–13] reported a specific microwave effect which cannot be reduced entirely to macroscopic heating. The possible pathways by which microwaves could affect chemical and biochemical processes in- clude orientational effects [1, 2] although only a few works [8–10, 14] addressed these issues so far. In this study we apply the microwave field with a well-defined geometry and focus on such microwave- induced protein response that carries information about protein orientation. This could be the polarization of the fluorescence emission which is characterized by the fluorescence anisotropy: r = I s − GI p I s +2GI p . (1) Here I s ,I p are the intensities of the emitted light with the s- and p-polarizations and G is a cali- bration factor [15]. The maximum value of the fluorescence anisotropy, achieved for polarized light excitation in the ensemble of immobile molecules with parallel absorption and emission transition moments, is r 0 =0.4. The partial loss of the fluorescence anisotropy, due to random fluorophore rotation in liquid, is described by the Perrin equation r = r 0 1+ τ F /τ D (2) where τ F is the fluorescence lifetime, τ D = ηV/k B T is the rotational correlation time, η is the solvent viscosity and V is the hydrodynamic volume of the fluorescent molecule. We compared the fluorescence emission spectra I s (λ),I p (λ) of the aqueous solution of the En- hanced Green Fluorescent Protein (EGFP) under continuous microwave irradiation and under conventional heating. The EGFP [16] has τ D = 13–18 nsec, τ F =2.6–3 nsec [17–19], and r 0 =0.38– 0.39 [15]. Figure 1 shows our optical setup. A glass pipette with an inner diameter of 1.2 mm and an outer diameter of 1.5 mm contained ∼ 5 μl of the 0.84-mg/ml solution of EGFP in Tris buffer with pH 8.0 [11]. The pipette was mounted in the center of a TE 011 microwave cavity operating at 9.5GHz and made of 5-mm thick brass (Fig. 2). The fluorescence was excited by a linearly polarized 488-nm Argon laser and was measured by a photomultiplier followed by a photon counter. To determine