Biochimica et Biophysica Acta, 491 (1977) 149-154

© Elsevier/North-Holland Biomedical Press BBA 37610 L U M I N E S C E N C E OF P H E N Y L A L A N I N E D I S M U T A S E F R O M G R E E N PEA




E. A. PERMYAKOV ~, E. A. BURSTEIN a. Y. SAWADAb and I. YAMAZAKI ~ °Institute of Biological Physics, U.S.S.R. Academy of Sciences, 142292 Puschino, Moscow region (U.S.S.R.) and bBiophysical Division, Research Institute of Applied Electricity, Hokkaido University, Sapporo (Japan)

(Received September 22nd, 1976)

SUMMARY The fluorescence properties of phenylalanine residues in superoxide dismutase from green pea have been studied. The fluorescence spectra of superoxide dismutase have a fine vibrational structure. The fluorescence quantum yield of intact protein in pH region from 5 to 10 is essentially constant and very low (around 1.1 ~). The thermal denaturation of the protein takes place at the temperatures above 70 °C.

INTRODUCTION The intrinsic fluorescence of proteins is due to the aromatic amino acid residues of phenylalanine, tyrosine and tryptophan. However, the values of extinction coefficients and fluorescence quantum yields of their aromatic or heterocyclic moieties are very distinct. Proteins containing the three (class B proteins [1]) sorts of aromatic residues show almost exclusively tryptophan fluorescence although it is possible to observe a minor contribution from tyrosine. Due to a very low value of extinction coefficient for phenylalanine (EM = 200) and very efficient energy transfer to Tyr and Trp its fluorescence is completely absent in the emission spectra of such proteins. In the absence of tryptophan (class A proteins [1 ]), proteins demonstrate the fluorescence of their tyrosines without any traces of phenylalanine emission. Only recently the fluorescence of phenylalanine in proteins devoid of tyrosine and tryptophan (class C proteins [2]; parvalbumins from muscles of fishes [3] and horse hepatocuprein [4]) has been registered [2, 4]. In the present communication we report the luminescence properties of phenylalanine residues in superoxide dismutase from green pea. This protein contains 9 phenylalanines and neither tyrosine nor tryptophan and has a molecular weight of 31 500 [5]. Superoxide dismutase is a metalloprotein and includes in its active site two Cu(II) and two Zn(II) atoms. MATERIALS AND METHODS All chemicals used were of grade "chemically pure". Superoxide dismutase was isolated from green peas as described previously [5]. The protein has been stored in

150 buffer solution (0.01 M K2PO 4, 0.05 M KC1, pH 7.8) in concentration 35 mg/ml at 0 to 4 °C for several months. The concentration of the protein during the measurements was 17 mg/ml. Spectrophotometric measurements were performed with Specord UV-VIS recording spectrophotometer (Karl Zeiss, Jena, G.D.R.). Fluorescence spectra at 293 °K were recorded with a recording instrument built in the Institute of Biological Physics, Acad. Sci. U.S.S.R. (Puschino). The excitation of fluorescence was produced by the light of mercury line triplet at 265 nm, isolated by a grating monochromator from the emission of the superhigh pressure lamp SWD-120. The fluorescence light from the front surface of the quartz cell (I0 × 10 × 40 ram) was focused on the entrance slit of the quartz prism monochromator SF-4. The quartz photomultiplier FEU-39 (Sb-Cs cathode) was used as a detector behind the exit slit of this monochromator. The photocurrent was registered on a recording potentiometer EPP-09M3. The hollow cell holder allowed the thermostabilization within the cell by means of water flowing from an ultrathermostat NBE U-3 (VEB Prfifgerfite, Medingen, G.D.R.). The temperature was measured directly in the cell using a semiconductor microthermistor MT-54. The temperature dependence of fluorescence intensity (the thermal quenching curve) was directly registered on an X-Y recorder PDS-021, where the signal from photomultiplier was applied to the X-input and that from the thermistor bridgecircuit to the Y-input. Since during heating the fluorescence spectrum did not change in shape and position, the intensity at any wavelength was proportional to the fluorescence quantum yield value. The rate of temperature change was about 10 °C/min. All the spectra were corrected for spectral sensitivity of the monochromatorphotomultiplier system. Intensities in the corrected spectra are proportional to the number of photons per one nm. The spectral slit width of the recording monochromator had the values from 1.1 nm (at 270 nm) to 1.6 nm (at 310 nm). The fluorescence spectra of the protein were corrected for screening and reabsorbing inner filter effects. The expression derived earlier [6] was used as a correction factor at every fluorescence wavelength (2): 1 -- Tp

w (2) -- I --


Dp + D~ + D r



where T and D are transparency and absorbance (T = 10 -°) respectively. Subscripts p and e refer to protein and screening agents at excitation wavelengths; r refers to reabsorbing agent at the wavelength of fluorescence 2. The reproducibility of intensities registered was not worse than + 5 ~ . The fluorescence quantum yield values were estimated by comparing the areas under the fluorescence spectra of the protein solution studied and those of aqueous phenylalanine solution of the same optical densities at the excitation wavelength. The quantum yield of phenylalanine fluorescence was assumed to be 0.038, according to Teale and Weber [7]. RESULTS The fluorescence spectrum of 5.4-10 -4 M intact superoxide dismutase in

151 ,3 100.









Fig. 1. Fluorescence spectra of aqueous phenylalanine (1), hake parvalbumin, pH 7.6 (2), and green pea superoxide dismutase, pH 7.8 (3). 20 °C; excitation at 265 nm.

phosphate buffer (0.01 M KzPO4; 0.05 M KC1, pH 7.8) at 293 °K excited at 265 nm has a shape very similar to that of aqueous phenylalanine, but the protein spectrum is shifted to longer wavelengths by 1-2 nm (see Fig. 1). The pH dependence of protein fluorescence quantum yield is shown in Fig. 2. In the pH range from around 5 to 10 the fluorescence quantum yield is essentially constant and very low (around 1.1 ~). The rises of fluorescence quantum yield at pH below 5 and above 10 seem to be due to the acidic and alkaline denaturation of the protein. The rise in the acidic region is accompanied by an essential decrease of the strong background absorbtion band, which probably is due to copper and zinc complexes (Fig. 3). This seems to be a result of the release of these metal atoms from the protein ligands during denaturative unfolding. Since only phenylalanyls absorb the light at 250-270 nm at pH 2.8 the measurement of fluorescence quantum yield of superoxide dismutase was most correct at this pH value. Because of the independence of spectral shape and position on pH q 0.06, O


0.05 0.O4 0.03.



Fig. 2. pH dependence of fluorescence quantum yield of green pea superoxide dismutase (1) and hake parvalbumin (2) 20 °C," excitation at 265 nm.

152 D

0.6. ~ 0.5'






\ 250 260 270nm280 290


Fig. 3. Absorption spectra of superoxide dismutase at pH 7.8 (curve 1) and 2.8 (curve 2). 20 °C.

value the maximal fluorescence intensity at 282 nm, corrected by screening effect of the background absorbtion band according to Eqn. 1 was assumed to be proportional to the quantum yield. That allowed us to calculate and to depict the quantum yieldp H plot (Fig. 2). Fig. 4 shows the temperature dependence of the fluorescence quantum yield for superoxide dismutase in a range from 5 to 80 °C. When temperature rises from 5 to 70 °C, the fluorescence yield monotonously decreases only by about 5 %. Above 70 °C the fluorescence quantum yield value shows a three-fold rise in the temperature range around 7 °C (the midpoint is 75 °C). This effect seems to be due to the thermal



0.031 0"02l

o.:l 0

20 40 60


Fig. 4. The thermal quenching of the fluorescence of superoxide dismutase (curve 1) and hake parvalbumin (curve 2). Excitation at 265 nm; registration at 282 nm.

153 denaturation process. The post (approx. 4 min) recooling of the solution does not result in any restoration of low quantum yield values intrinsic to the native protein. However some hysteresis feature at approx. 61 °C is observed. DISCUSSION It is of interest to compare the fluorescence properties of superoxide dismutase with those of parvalbumins which also contain no tyrosine and tryptophan residues and show the phenylalanine fluorescence [2]. First of all, the fluorescence spectra of both superoxide dismutase and parvalbumins have identically well resolved vibrational structure peculiar to phenylalanine chromophore. The fluorescence spectra of these proteins are slightly shifted to the long wavelength region in comparison with the aqueous phenylalanine spectrum. Taking into consideration the similar shifts of the absorption spectra of the proteins, one can assume that the fluorescence spectral shifts of phenylalanine residues in proteins are due in a great extent to the intramolecular inductive effects of substituent in benzene ring. The solvent effects seem to be negligible in this case since the fluorescence spectra of low-molecular weight phenylalanine derivatives are practically insensitive to the solvent replacement [8]. The fluorescence quantum yield of the native superoxide dismutase (1.1 ~) is much lower than that of aqueous phenylalanine (3.8 ~), while the quantum yield values of parvalbumins in neutral pH range are as high as 5-25 ~o. The denaturation of superoxide dismutase by acid, alkali or heating leads to the sharp rise of its quantum yield up to values near peculiar for the aqueous phenylalanine. The acidic and alkaline denaturation of parvalbumins results in a decrease of their fluorescence quantum yield up to almost the same value. It seems possible that in proteins of both kinds in their native states the phenylalanine residues are located in the interior of the protein molecule, but some of the phenylalanine residues in superoxide dismutase are in contact with copper or zinc ions, which leads to very strong quenching of the phenylalanyl fluorescence. The denaturation of these proteins results in increase of the accessibility of their phenylalanine residues to a solvent and in release of metal ions from protein ligands. Consequently, the environment of phenylalanine residues in both kinds of proteins becomes almost the same. Heating to denaturation causes a decrease of fluorescence quantum yield for both superoxide dismutase and parvalbumins, but in the case of parvalbumins this decrease is much more pronounced. Such a weak temperature dependence of the native superoxide dismutase quantum yield is very unusual for proteins. Since the quenching of the fluorescence of protein chromophores is limited by the collision frequency with nearby quenching groups, the thermal quenching curve reflects the temperature dependence of the structural mobility of the chromophore environment in protein [9, 10]. Therefore it seems possible that the absence of the thermal quenching of native superoxide dismutase is due to unusually high rigidity of structure in this protein. The structure ofparvalbumin seems to be not so rigid and the thermal quenching of its fluorescence is rather well pronounced (Fig. 4). REFERENCES 1 Teale, F. W. J. (1960) Biochem. J. 76, 381-390

154 2 Burstein, E. A., Permyakov, E. A., Emelyenenko, V. I., Bushueva, T. L. and Pechere, J. F. (1975) Biochim. Biophys. Acta 400, 1-16 3 Pechere, J.-F. (1968) Comp. Biochem. Physiol. 24, 289-295 4 Finazzi-Agro, A. (1974) FEBS Lett. 39, 164-166 5 Sawada, Y., Ohyama, T. and Yamazaki, I. (1972) Biochim. Biophys. Acta 268, 305-312 6 Burstein, E. A. (1968) Biofizika 13, 433-437 7 Teale, F. W. J. and Weber, G. (1957) Biochem. J. 65, 476-482 8 Burstein, E. A. (1976) Luminescence of Protein Chromophores. Model Investigations, Viniti, Moscow 9 Bushueva, T. L., Busel, E. P. and Burstein, E. A. (1975) Stud. Biophys. 51, 173-182 10 Bushueva, T. L., Busel, E. P. and Burstein, E. A. (1975) Stud. Biophys. 52, 41-52

Luminescence of phenylalamine residues in superoxide dismutase from green pea.

Biochimica et Biophysica Acta, 491 (1977) 149-154 © Elsevier/North-Holland Biomedical Press BBA 37610 L U M I N E S C E N C E OF P H E N Y L A L A N...
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