Spectroscopic properties and stability of hemocyanins

SPECTROCHIMICA ACTA PART

A

Spectrochimica Acta Part A 53 (1997) 471-478

ELSEVIER

Spectroscopic properties and stability of hemocyanins Rumijana

‘Department

Hristovaa,

Pavlina Dolashka”, Stanka Stoevab, Wolfgang Benedetto Salvato”, Nicolay Geneva**

“Institute of‘ Organic Chemistry, of Physical Biochemistry at the Institute ‘Department

Voelterb,

Bulgarian Academy of Sciences, Sofa 1040, Bulgaria of Physiological Chemistry, University of’ Tiibingen, Hoppe-Sevler-Stra,& 72076 Tiibingen, Germany of Biology, University of Padova, Padova, Ita1.v

4,

Received 16 August 1996; accepted 17 August 1996

Abstract

The stability towards thermal and chemical(guanidinehydrochloride) denaturation of oxy- and apo-hemocyanins from the arthropodan organismsHomarus americanus, Maia squinado, Palinurus vulgaris and Carcinus maenas aswell as from the molluscsRapana thomasiana and Viviparus ater have beeninvestigatedby fluorescencespectroscopyand circular dichroism. The H. americanus hemocyaninshowedan extreme thermostability in comparisonto the other investigatedhemocyanins.The critical temperatureof deviation from linearity (r,) of the Arrhenius plot, ln( Q ’ - 1) vs. l/T, where Q is the protein quantum yield of fluorescence,wascalculatedto be 87°C for this respiratory protein. The T,-values for the other hemocyaninsrange between63 and 76°C. The respectiveactivation energiesfor the radiationlessthermal deactivation of the excited indole chromophoreswere calculated to be 37.0-50.5 kJ mol--‘. Guanidinehydrochloride is an efficient denaturant for hemocyanins.The protein unfolding wasmonitored by circular dichroism.The free energy of stabilization in water, AGE’O, at 25°C and pH 7.5, was calculatedto be in the range 8.0-21.6 kJ mol-‘. The highest AGEZo-values were calculated for the Rapana thomasiana hemocyanin. Upon excitation at 295 or 280 nm the fluorescenceemissionof the investigatedhemocyaninsis dominated by ‘buried’ tryptophyl chromophores.The removal of the copper-dioxygensystemfrom the active siteled to 3.8-7.9-fold increase of the protein fluorescencequantum yield and to a red shift of the emissionmaximum position. Evidently, the tryptophyl fluorescenceis significantly quenchedin the oxy-hemocyanins.0 1997Elsevier ScienceB.V. Keywords:

Activation energy: Circular dichroism; Denaturation; Fluorescence;Hemocyanin; Invertebrate: Stability

1. Introduction Abbreviations: Hc, hemocyanin: GnHCl, guanidine hydrochloride; AGElo, free energy of stabilization in water; T,, the temperature at which the Arrhenius plot deviate from linearity. * Corresponding author. Tel.: + 359 2 7136677; fax: + 359 2 700225.

Hemocyanins (Hcs) are respiratory proteins freely dissolved in the hemolymph of invertebrates from the two phyla, Arthropoda and Mollusca [l]. They contain a binuclear copper active site which binds dioxygen reversibly. Each of the two metal

1386-1425/97/$17.00 0 1997 Elsevier Science B.V. All rights reserved. PIIS1386-1425(96)01837-9

ions is coordinated to three histidyl residues. The copper ions are inequivalent with respect to many reactions and can be removed sequentially from the active site [2]. The molecular architecture of Hcs from the two phyla is different. The subunits of arthropodan Hcs form hexamers or multiples of hexamers [3]. Each subunit of 67-90 kDamolecular mass contains one dioxygen-binding site [4]. The crystal structure of two respiratory proteins from the arthropods Panulirus interruptus [S] and Limulus polyphemus [6] have been resolved at 0.320 and 0.2 18 nm, respectively. In electron micrographs molluscan Hcs appear as cylinders with an external diameter of 300-350 A and height of 350-380 A. 10, 20 or more subunits build up the cylindrical aggregates with a molecular mass of up to 43.4 x lo6 Da. Each polypeptide chain is composed of seven or eight domains; each domain contains a single binuclear active site [7]. The quaternary structure of the Hc from the gastropod Megatura crenulata (the keyhole limpet Hc) has been recently determined and the domain structure of the constituting subunits has been analysed too [8]. This protein is an immune stimulant and it was used for immunotherapy of cancer [9, lo]. Three-dimensional reconstructions of molluscan Hcs from different infraorders have been performed by the group of J.N. Lamy [l 1- 141. Although the functional and some structural properties of Hcs have been intensively studied, little is known about the stability of these respiratory proteins. Only a few papers on this problem have been published. Thus, the urea denaturation of the Carcinus maenas Hc was investigated and it was shown that increasing concentrations of this reagent disrupted the secondary and tertiary protein structures [ 151. The thermostability of Hcs from Octopus vulgaris [ 161, Palinurus vulgaris [ 171 and Eurypelma caltjbrnicum [18] was also studied by different methods. This study describes spectroscopic properties and the conformational stability towards heat and guanidine hydrochloride (GnHCl) induced denaturation of arthropodan (crustacean) Hcs from the infraorders Astacidea (Homarus americanus), Brachyura (Carcinus maenas, Maia squinado) and Palinura (Palinurus vulgaris) as well as of molluscan (gastropodan) Hcs from Rapana thomasiana grosse

(marine snail) and Viviparus ater (a small terrestrial snail). The spectral characteristics are tentativelyexplained with peculiarities of the protein structure. Investigations with native and apo-hemocyanins were performed in order to study the role of the copper-dioxygen system for the spectral parameters and for the stabilization of the protein structure in solution. All experiments were performed using the same conditions and instruments allowing direct comparing of the obtained results. According to our knowledge, the investigations described in this paper are the first systematic and comparative study on the stability of hemocyanins belonging to differentphyla.

2. Experimental 2.1. Hemocyanins

The hemocyanins from Homarus americanus, Carcinus maenas, Maia squinado, Palinurus vulgaris and Viviparus ater were purified by the method described in [ 161. The Rapana thomasiana grosse hemocyanin was isolated and purified as described in [19]. The proteins were stored at - 20°C in the presence of 18% sucrose. Before use they were dialyzed against a 50 mM Tris/HCl buffer, pH.7.5, containing 5 mM CaCl,. Apo-Hcs (hemocyanins in which the copper-dioxygen system is removed) were obtained by dialysis against 25 mM KCN [20]. 2.2. Spectroscopic

measurements

Fluorescence spectra were recorded on a PerkinElmer model LS5 spectrofluorimeter equipped with a thermostated cell compartment and a Data Station, model 3600. The absorbance of the protein solutions was lower than 0.05 at the excitation wavelength to avoid inner filter effects. Fluorescence emission spectra were recorded between 20 and 95°C. The samples were equilibrated at each temperature for 10 min before the measurements. Fluorescence quantum yields were calculated by the equation [21]:

where Q,, Fx and A, are the emission quantum yield, the area of the emission spectrum and the

R. Hristova et al. / Spectrochimica Acta Part A 53 (1997) 471-478

optical density at the excitation wavelength, respectively, for the protein sample, and Q,, Fs, and A,, are the same parameters for the reference standard. N-Acetyltryptophan amide (Ac-TrpNH,) with a quantum yield of 0.13 [22] was used as a standard. Circular dichroism measurements were performed on a Roussel Jouan Dichrographe III instrument. Protein samples in 50 mM Tris/HCl buffer, pH 7.5, containing 5 mM CaCL, were incubated at 25°C with GnHCl at different concentrations and CD spectra were recorded between 200 and 250 nm. The attainment of equilibrium at each concentration of the denaturant was confirmed with the constancy of the ellipticity at 222 nm. The data about the temperature dependence of the fluorescence quantum yield were analysed according to the equation [23]: ln(Q- ‘-l)=lnf;/Kf-E,/R.l/T where Q is the fluorescence quantum yield, J;. is the frequency factor for the nonradiative deactivation processes, Kr is the temperature independent rate constant for emission of fluorescence [23], E, is the apparent activation energy; R is the gas constant and T is the absolute temperature. Protein concentrations were measured spectrophotometrically at 278 nm using absorption coefficients Eiq& = 13.4 for the H. americanus Hc [24]; 13.8 for the P. oulgaris Hc [25]; 12.4 for the C. maenas Hc [ 151; 13.5 for the M. squinado Hc; 13.8 for the R. thomasiana Hc [19] and 13.6 for the V. ater Hc. Absorption spectra were recorded with a Shimadzu spectrophotometer, model 3000. 2.3. Cdculution

oj’ the free energy of stabilization

The free energy of guanidine denaturation, AC,, was estimated from the equation: AG,=

-RTlnK

where K is the equilibrium constant of the denaturation process. K was calculated for each denaturant concentration according to the equation:

K = V%,, - Pl,,/C[%

- [@Lx)

313

where [a],,, is the observed ellipticity at 222 nm at different concentrations of GnHCl, and [O], and [O], are the ellipticities at the same wavelength for the folded (N) and unfolded (D) conformations of the proteins, respectively. The value for the free energy of stabilization in the absence of denaturant, AG$O, was obtained by linear extrapolation of AG, to zero molar denaturant [26-281.

3. Results and discussion

Fluorescence spectroscopy and circular dichroism are sensitive methods for studying protein conformation in solution and changes in conformation. Both methods can be used to study folding-unfolding processes in proteins. Table 1 summarizes fluorescence parameters of Hcs from arthropodan and molluscan organisms. Experiments were performed on native and apo-proteins in order to study the effect of copper-dioxygen system on the fluorescence properties of the investigated respiratory proteins. Hcs contain tyrosyl and tryptophyl fluorophores. Upon excitation at 295 nm, where the light is specifically (93%) absorbed by the tryptophyl residues or at 280 nm, where both phenol and indole groups absorb, the native and apo-Hcs showed an emission maximum position, rl,,,, in the region 321-331 nm which is typical for buried tryptophyl side chains in non-polar environment [29,30]. Evidently, the fluorescence of the both forms is dominated by deeply buried tryptophans. This fact makes fluorescence spectroscopy a suitable method for investigating unfolding reactions in hemocyanins, because the emission characteristics of these proteins will be considerably changed during the denaturation process due to the gradual ‘exposure’ of the indole chromophores to the water solvent. The differences in /i,,, reflect differences in the local environment of the emitting fluorophores. Fluorescence emission spectra in the temperature interval 19-85’C, after excitation at 295 nm of apo-Hc from M. squinado at pH 7.5 are reported in Fig. 1. A decrease of the fluorescence intensity upon raising the temperature was observed. The quenching is due to the thermal acti-

Table

I

Fluorescence

parameters

of hemocyanins A,,,

Hemocyanins

25°C) Arthropods Homurus form ) Honrarus

from

(i,,

= 295

(nm)

arthropodan nm.

and molluscan

organisms

I,,,, (icy = 280 nm. 25°C) (nm)

@anturn CP)

americanus

(oxy-

324 i I

322 * I

0.01’

ctmericunits

(ape-

331 + 1

3265

I

0.085

* I 325 I I 326i I

323* 324kl

I

321 f 1

0.013

331+

1

331*1

0.082

324*

1

325k

I

0.014

327f

I

326f

I

327 + I 330* I

328k

I

yield

E,, (kJ mol

‘/

7; (“i‘)

Xl.5

81

48.3

76

37.7

67

0.110

33.7

63

0.012“

37.0

65

45.7

74

form)

/M~icc syuinado (oxy-form) Muiu squinado (apo-form) Palinurus vulgaris (oxyform) Palinurus vulgaris (apoform) Car&us maenas (oxyform) Curcitzus mamas (apoform) Molluscs C’ioiparus ater (oxy-form) C’iciparu.5 ater (apo-form) Rapana thomasiarta (oxyform) Rupana thomasiana (apoform)

323

33011

0.074"

0.012

325*

I

321 i I

329+

1

326k

1

0.049

The optical absorbance of the solutions was lower than 0.05 at the excitation critical temperature of deviation from linearity of the Arrhenius plot [(In Q-j thermal deactivation of the excited protein chromophores. ” Data from [37].

vation of intramolecular collisions between excited indole groups and neighboring groups [31]. There was not a peak for tyrosyl fluorescence which can be expected at 303-304 nm [32]. The absence of a ‘tyrosine’ peak in the spectra can be explained with an efficient radiationless energy transfer from phenol to indole groups. Electronic excitation energy transfer occurs between a donor and acceptor over distances as large as 100 A [33]. Crystallographic investigations at 3.2 A resolution [34] showed that the arthropodan hemocyanin functional subunit is an ellipsoid of dimensions 48 x 55 x 80 A. The X-ray models [5,6,34] demonstrated many possibilities for an efficient Tyr-to-Trp energy transfer. The functional unit of the molluscan Hcs is smaller. Another explanation for the absence of tyrosyl fluorescence is a quenching of the phenol emission by a specific environment. The shift of R,,, under excitation at

-~

0.021 0.079

wavelength, - I) vs. l/q.

avoiding inner filter E, is the activation

effects. energy

r, is the from the

280 nm is an indication of the heterogeneity of tryptophan environment. The fluorescence emission quantum yields (Q) of all native Hcs are very low (Table 1). This can be explained with a quenching effect of the specific environment of the emitting tryptophans. The values of /i,,, and Q for the C. maenas Hc are in agreement with those reported in [15]. The copper-dioxygen system removal caused 3%7.9-fold increase of the quantum yield and a red shift of the emission maximum position (Table 1). Circular dichroism spectra of the native and apo-Hcs were essentially identical, suggesting a closely similar folding of the polypeptide chains of the two forms. The CD data showed that little or no conformational changes occur in the polypeptide backbone upon the removal of the copper-dioxygen system and the fluorescence quenching can not be explained with structural

R. Hristova

et al. / Spectrochimica

changes. Evidently, the system mentioned above quenches the emission of tryptophans located in a close proximity of the active site. Upon the copper removal, the quenched residues, which are the main contributors to the total protein emission of the apo-Hcs, become fluorescent. Between 73 and 87% of the overall emission is associated with these fluorophores. The functional units of the arthropodan and molluscan Hcs contain 7-8 tryptophyl residues [35]. The observed quenching can be explained by a ‘heavy atom’ effect and a ‘paramagnetic ion’ effects due to the oxidized copper [36]. The character of the emission is informative for a hydrophobic environment of the respective active sites. The study of Hc conformational stability is of fundamental interest to our understanding of the structure-function relationships in these dioxygen carriers. We have examined the stability of Hcs as a function of temperature in the range from 20 to 95°C by fluorescence emission spectroscopy, in the presence of physiological concentration of calcium ions. The temperature dependence of the fluorescence quantum yield was investigated only for the apo-Hcs due to the effect of the bound to the native protein dioxygen, which is liberated at higher temperatures. The thermal denaturation of the Hcs, i.e. the unfolding process that takes place after passing the critical temperature, was irreversible and for this reason equilibrium thermody-

Fig. 1. Fluorescence emission spectra after excitation at 295 nm of apo-hemocyanin from Muia squinado in the temperature interval from 19 (the upper curve) to 85 (the lowest curve)“C at pH 7.5.

Acta Part

A 53 (1997)

471-478

375

;

1 2.7

2.9

3.1

3.3

,/T x lO’(‘K-‘)

Fig. 2. Thermal dependence of the tryptophyl fluorescence of the Homarus americanus apo-hemocyanin in 0.05 M phosphate buffer, pH 7.5. The fluorescence quantium yields were determined using Ac-Trp-NH, as a standard.

namic parameters were not determined. This type of behaviour was shown by other authors for the Palinurus vulgaris Hc [17] and other proteins [3739]. The irreversibility of denaturation can be explained with the inclination of the huge protein molecules to aggregate. We use T,-values, the critical temperature for deviation of the Arrhenius plot, ln(Q - ’ - 1) vs. l/T, from the linearity. to characterize the thermostability of the investigated respiratory proteins. The deviation indicates that at temperature higher than i”, the protein undergoes denaturation. A typical Arrhenius plot for the H. americanus Hc is shown in Fig. 2; similar plots were obtained for the other Hcs. Table 1 summarizes data about the activation energy for the thermal deactivation of the excited protein fluorophores, E,- and T,-values. E, ranges between 33.7 and 50.5 kJ mol ~ ‘, As can be seen, the H. americanus Hc shows an extreme thermostability. This respiratory protein was conformationally stable at temperatures up to 87°C and only some monotonical decrease of the emission with the increase of temperature, characteristic for the intrinsic temperature dependence of the tryptophan emission but not for conformational changes, was observed. It was shown [18] that the Hc from the tarantula Eurypelma californicum, an organism living in areas of large temperature lluctuation of more than 70°C between day and night, is conformationally stable and fully functioning at temperatures up to 90°C. This was explained with

an effect of the specific environment created by the desert in which the organism is living. HOWever, the North American lobster H. clnwriccmu inhabiting the Atlantic Ocean, is not living under such extreme environmental conditions, but its Hc also possessesa high thermostability. The extreme thermostability of the two Hcs can be explained with their high oligomerization and with an increased stability of individual polypeptide chains. The hexamers of the E. calijbrnicur~z Hc form dodecamers that dimerize to the native 24-merit aggregate containing seven different types of subunits [18]. The H. mzericanus Hc has a dodecameric structure organized from two basic hexameric units [40]; the aggregates are composed of six major and two minor electrophoretically separable polypeptide chains [41]. The different subunits may have different denaturation kinetics. The temperature of deviation, T,, for the other Hcs is lower (Table 1) indicating differences in the thermostability of the investigated proteins. They began to denature in the temperature interval from 63 to 76°C. Our data about the P. vulgaris Hc are consistent with those from the differential scanning calorimetry measurements reported in [17]. It should be mentioned that even after heating at 90-95°C the spectral maximum position was lessthan 342 nm which is typical for ‘buried’ tryptophans. Probably, the hemocyanin polypeptide chains are not completely unfolded at these conditions. The unfolding reactions of Hcs in the presence of GnHCl were followed by CD-spectroscopy. We have chosen [OlZZ2 to follow the unfolding process. Chemical denaturation experiments were performed on oxy- and apo-proteins. The native conformation was stable up to 1 M GnHCl. At increasing denaturant concentrations a general decrease in the negative ellipticity was observed and the respiratory proteins gradually denatured. A curve demonstrating the change of ellipticity at 222 nm during the denaturation of the native M. squirmdo HC is shown in Fig. 3. Similar curves were obtained for the other Hcs. From 1 to 4 M GnHCl an extensive disruption of the hemocyanin structure occurs. The midpoints of the transition curves for the native proteins are in the region 2.9-3.7 M guanidine; those for the apo-forms are

between 2.4 and 2.9 M GnHCl. The transition midpoints for the apo-Hcs were shifted toward the lower denaturant concentration; a shift equal to approximately 1 M of guanidine was observed. It can be concluded that the active site copper ions are not critical but important for the maintenance of the native conformation. The CD spectra showed a general unfolding of the native Hcs in 6 M GnHCl and of the apoproteins in 5 M concentration of the reagent. This finding confirms that the apo-forms are more sensitive to the action of the denaturant. No ordered structure was observed in 8 M GnHCl and the CD spectra were typical for unfolded proteins. This is in contrast to the high stability of the C. nzaenas hemocyanin aggregates towards

I

, 2

I

I

I

4

6

0

[Gn. WI].

(Mj

Fig. 3. Guanidine hydrochloride-induced denaturation at pH 1.5 and 25°C of the native Maia squinado hemocyanin (the lower panel of the figure). The upper panel shows the free energy change, AG,, for the unfolding reaction of the same protein as a function of GnHCl concentration.

R. Hristoaa Table 2 Free energy hemocyanins

et al. / Spectrochimica

of stabilization in water at pH 7.5 and 25°C from arthropodan and molluscan organisms kJ mall

of

Acta

Part

A 53 (1997)

471-478

471

proteins. The stability of Hcs towards guanidine denaturation is relatively low compared with the majority of the globular proteins [42-441.

Hemocyanins

AGgI”

’

Arthropods Homarus americama (oxy-form) Homarus amrricanus (apo-form) Maia squinado (oxy-form) Muia squinado (apo-form) Palitirrrus uulgaris (oxy-form) Palinwus u&ark (apo-form) Car&us maenas (oxy-form) C‘arcinus ~n0e11~l.s (apo-form)

13.5 12.5 12.6 11.4 12.2 8.0 11.4 10.3

Acknowledgements

Molluscs L’ioipurus ater (oxy-form) Viuiparus uter (apo-form) Rupana thomasiurla (oxy-form) Rupana thomasiana (apo-form)

12.6 10.5 21.6” 18.0”

References

We express our gratitute to the VolkswagenStiftung (Hannover, Germany) for financial support by the research grant No I/70 524.

[l] [2]

The unfolding reactions of the investigated oxygen-transporting proteins, in the presence of different concentrations of guanidine hydrochloride, were followed by CD spectroscopy. :’ Data from [37].

[3] [4] [5]

urea: even in 8 M urea some ordered structure is still present [16]. The renaturation of Hcs was performed by lowering slowly the guanidine concentration. The CD spectra, recorded after removal of the denaturant, suggest between 53 and 79% recovery of secondary structure and were characteristic for higly ordered proteins. Only in the case of the V. afrr Hc the degree of renaturation was 10%. The measured dichroic spectra can be considered as an approximation to the true equilibrium spectra. GnHCl is a commonly used denaturant to determine the free energy of protein stabilization in water, AGz20 [28]. At 25°C and pH 7.5 this parameter was calculated to be in the region 8.0-21.6 kJ mol ~ ’ for the investigated Hcs (Table 2). The values for the native proteins were higher than those for the apo-forms. The active site copper ions stabilize additionally the native Hc conformation in solution by 1.0-4.2 kJ mol1. The investigated Hcs, except that from Rapana thornasiana, show only limited differences in the stability towards GnHCl. The experiments demonstrated that GnHCl is an effective denaturation reagent for these oxygen-transporting

[6]

[7]

K.E. van Holde and K.1. Miller, Q. Rev. Biophys.. 15 (1982) I. M. Beltramini, F. Ricchelli and B. Salvato, Inorg. Chim. Acta, 92 (1984) 209. J. Markl, Biol. Bull., 171 (1986) 90. T.T. Herskovits, Comp. Biochem. Physiol. B, 91 (1988) 597. A. Volbeda and W.G.J. Hol. J. Mol. Bioi., 209 ( I9891 249. B. Hazes, K.A. Magnus, C. Bonaventura, J. Bonaventura, 2. Dauter, K.H. Kalk and W.G.J. Hol, Protein Sci.. 7 (1993) 597. B. Salvato and M. Beltramini, Life Chem. Rep., 8 ( 1990)

[8] W. Gebauer, J.R. Harris, H. Heid, M. Siiling, R. Hillenbrand, S. Sohngen, A. Wegener-Strake and J. Markl, zoology, 98 (1994) 51. [9] CD. Jurinic, U. Engelmann, J. Gasch and K.F. Klippel. J. Urol.. 139 (1988) 723. [IO] K.H. Bichler, S. Kleinknecht and W.L. Strohmaier, IJrol. Int.. 45 (1990) 269. [ll] 0. Lambert, N. Boisset, P. Penczek, J. Lamy, J.-C. Taveau, J. Frank and J.N. Lamy, J. Mol. Biol.. 738 (1994) 75. [12] 0. Lambert. N. Boisset, J.-C. Taveau and J.N. Lamy. J. Mol. Biol., 244 (1994) 640. [13] 0. Lambert, N. Boisset. J.-C. Taveau and J.N. Lamy. Arch. Biochem. Biophys., 316 (1995) 950. [14] 0. Lambert, J.-C. Taveau, N. Boisset and J.N. Lamy, Arch. Biochem. Biophys., 319 (1995) 231. [15] F. Ricchelli, B. Salvato, B. Filippi and G. Jori, Arch. Biochem. Biophys.. 202 (1980) 277. [16] F. Ricchelli, G. Jori, L. Tallandini, P. Zatta, M. Beltramini and B. Salvato, Arch. Biochem. Biophys.. 235 (1984) 461. [17] M. Guzman-Casado. A. Parodi-Morreale, P.L. Mateo and J.M. Sanchez-Ruiz, Eur. J. Biochem.. 188 (1990) 181. [IS] R. Sterner, T. Vogl, H.-J. Hinz. F. Penz. R. Hoff. R. Fall and H. Decker, FEBS Lett.. 364 (1995) 9.

[19] R. Boteva. S. Severov, N. Genov, M. Beltramini. B. Filippi, F. Ricchelli. L. Tallandini. M.M. Pallhuber. G. Tognon and B. Salvato, Comp. Biochem. Physiol., IOOB (1991) 493. and F. Ghiretti. BioWI B. Salvato. A. Ghiretti-Magaldi chemistry, 13 (1974) 4778. PII E.P. Kirby and R.F. Steiner, J. Biol. Chem.. 245 (1970) 6300. 10 (1971) 3254. m S.S. Lehrer, Biochemistry, ~231 E.P. Kirby and R.F. Steiner, J. Phys. Chem.. 74 (1970) 4480. R.C. San George and L.J. ErhunmwunP41 T.T. Herskovits, see, Biochemistry, 20 (1981) 2580. C. Nuzzolo and F. Ghiretti, BioWI A. Ghiretti-Magaldi, chemistry, 5 (1966) 1943. 131 (1986) 266. WI C.N. Pace, Methods Enzymol.. 27 (1988) PI M.M. Santoro and D.W. Bolen, Biochemistry, 8063. 32 (1993) WI L.M. Mayr and F.X. Schmid, Biochemistry, 7994. J., 76 ( 1960) 381, 1291 F.W.J. Teale, Biochem. N.S. Vedenkina and M.N. Ivkova, Pho[301 E.A. Burstein. tochem. Photobiol.. 18 (1973) 263. V. Yarmolenko. E.A. Burstein and C. [311 E.A. Permyakov, Gerday. Biophys. Chem., I5 (1982) 19.

Biochcm. J 7-1.x I IYS7i [391 J.D.J. O’Neii and T. Hol’mann. 611. J. Photochem. Photobioi. B, 1’ (199)2) .i2?. [331 L. Matyus. Gaykema, W.G.J. Hol. J.M. Vrrcijken. Y.M. [341 W.P.J. Soeter. H.J. Bak and J.J. Bcintema. Nature, 309 (1984) 23. C. Nuzzolo and 1:. Ghirctti. BIOc351 A. Ghiretti-Magaldi. chemistry, 5 (1966) 1943. M. Beltramini, L. Flamigni and B. Salvato. 1361 F. Ricchelli, Biochemistry, 26 (1987) 6933. J.L. Lopez-Lacomba, M. Cortijo and [371 J.M. Sanchez-Ruiz, P.L. Mateo, Biochemistry, 27 (1988) 1648. J.L. Lopez-Lacomba, P.L. Mateo. M. [381 J.M. Sanchez-Ruiz. Vilanova, M.A. Serra and F.X. Aviles, Eur. J. Biochem.. 176 (1988) 225. B. Filippi, P. Dolashka, K.S. Wilson and Ch. [391 N. Genov, Betzel, Int. J. Peptide Protein Res., 45 (1995) 391. M.W. Russell and SE. Carberry, Bio1401 T.T. Herskovits, chemistry. 23 (1984) 1875. Biol. Bull.. 184 (1993) 105. [411 C.P. Mangum, V.V. Filimonov, P.L. Mateo ~421 A.R. Viguera, J.C. Martinez, and L. Serrano, Biochemistry. 33 (1994) 2142. S. Fahnestock, T. Lee. J. Orban and P. 1431 P. Alexander, Bryan, Biochemistry. 31 (1992) 3597. N. Genov, K. Parvanova, W. Voelter, M. [441 P. Doiashka. Geiger and S. Stoeva. Biochem. J.. 315 (1996) 139.