Photochemistry and Photobiology Vol. 44. No. 2 , pp. 159 - 167, 1986 Printed in Great Britain

0031 -8655186 $03 .00+0.00 Pergamon Journals Ltd

COMPARISON OF THE SPECTROSCOPIC PROPERTIES OF 4'-AMINO PHENYLALANINE AS AN UNPROTECTED

AMINO ACID AND AS A RESIDUE IN PEPTIDES ANDRZEJ JANKOWSKI~* and PIOTR DOBRYSZYCKI'

'Institute of Chemistry, Wroclaw University, Joliot-Curie 14, 50-383 Wroclaw, Poland and 'Institute of Organic and Physical Chemistry, Technical University, Wybrzeie Wyspianskiego 27, 50-370

WrocIaw. Poland

(Received 15 November 1985; accepted 3 March 1986)

Abstract-Ultraviolet absorption and fluorescence spectra of 4'-amino phenylalanine (Aphe) and of some peptides and esters containing Aphe, are characterized. Based on these results, the applicability of Aphe for fluorescence structural investigations of peptides is evaluated. The use of Aphe as an acceptor in fluorescence energy transfer measurements offers the possibility of facilitating the determination of donor fluorescence quantum yield (I$;) in the absence of energy transfer which usually turns out to be difficult in systems containing donor and acceptor moities. The Aphe residue in peptides also allows a better insight into intramolecular interactions.

INTRODUCTION

4'-Amino phenylalanine which hereafter is referred to as Aphei is an analog of naturally occurring amino acids. It can be linked with a peptide by an amide bond. Aphe has been used for peptide modification (Siemion et al., 1981; Schwyzer and Caviezel, 1971) and for intramolecular distance determination by spectrofluorimetric techniques (Jankowski et a l . , 1981). The aim of the present work is a better characterization of the spectroscopic properties of Aphe by comparison of its absorption and fluoresc- ence spectra with those of its analogs (aniline, tyrosine, phenylalanine) as well as of simple peptides and esters containing Aphe.

The following compounds were studied: (1) Aphe a s a n u n p r o t e c t e d a m i n o a c i d , ( 2 ) N - butyloxycarbonyl derivative of Aphe (BocAphe), (3) methyl ester of Aphe (ApheOMe), (4) Aphe-glycyl- glycine ethyl ester (APGGOEt), (5) analog of en- kephalin: Tyr-Gly-Gly-Aphe-Leu (A-enk).

In fluorescence energy transfer experiments Aphe as an acceptor is more convenient than traditionally used fluorescent probes due to the possibility of bleaching the acceptor by its protonation. This enables direct determination of donor fluorescence quantum yield in the absence of energy transfer (~$6).

MATERIALS AND METHODS

Preparations. A-enk and BocAphe were synthesized as described previously (Siemion el al . , 1981; Siemion et al., 1985, respectively). ApheOMe hydrochloride was obtained by nitration of D.I. phenylalanine followed by esterification

*To whom correspondence should be addressed. t A bbreviations: Aphe , 4'amino phenylalanine;

ApheH', protonated aphe; BocAphe, N-butyloxy carbonylo aphe; ApheOMe, methyl esther of aphe; APGGOEt, aphe-glycyl-glycine ethyl esther; A-enk, Amino-enkephalin (Tyr-Gly-Gly-Aphe-Leu).

(Deimer, 1974) and catalytic hydrogenation on palladium absorbed on charcoal. The final product recrystallized three times had a melting point of 182-184°C. APGGOEt was obtained as described by Stefanowicz (1984).

Chemicals. Aphe was purchased from Sigma Switzer- land. Sulphuric acid fuming + 65% SO3 was from Merck W. Germany. 'Other reagents were of standard analytical grade.

Spectrophotometric measurements. Spectrophotometric measurements were performed on a SPECORD UV-VIS (C. Zeiss - E. Germany). Aphe spectra were recorded using a Beckman UV 5240 instrument.

Maximum positions of absorption bands (V,,, in cm ') were determined with an accuracy of 80 cm-' and the values obtained were converted to wavelength (Amdx) in nm.

The change of molecular dipole moment of Aphe on its excitation (Ap*) was calculated from spectral shifts in aprotic solvents from Eq. 1 (Mataga and Kubota, 1970).

where h is Planck constant, D is dielectric constant, n is the refractive index of the solvent, a is the radius of cavity in the solvent produced by a solute molecule, derived from Onsager reaction field model.

Steady state fluorescence measurements. Fluorescence measurements were performed by means of Perkin-Elmer model 204 apparatus supplied with a recorder (Perkin- Elmer 56); the excitation wavelength was 250 nm and the optical density of the solution was 0.025-0.05. In every case contaminant fluorescence of the solvent was registered and subtracted from the sample spectrum. If the presence of Raman scattering peak was suspected the spectrum was checked by varying the excitation wavelength. Fluorescence quantum yields (4) were determined by comparing the area under the fluorescence spectrum with the corresponding areas of tryptophan (4 = 0.13, Schiller et al., 1977) and Aphe (I$ = 0.086) (Jankowski et al., 1981). The Aphe component in the fluorescence spectrum of A-enk, as well as the energy transfer efficiency were determined and the excitation spectra were corrected as described previously (Jankowski et al., 1981).

Spectrophotometric and spectrofluorimetric titrations. In the titration experiments the sample concentrations were

159

160 ANDRZEl JANKOWSKI and PIOTR DOBRYSZYCKI

varied accordingly since extinction coefficients of samples were markedly dependent on pH. Ionic strength of samples at low acidity was maintained constant. pH was measured using microelectrodes (Redox-Poland). High acidities of solutions in spectrofluorimetric titrations were obtained by addition of sulphuric acid. The values of Hammetts acidity function ( H , ) were determined from sulphuric acid concen- tration according to Hammett (1976). The acid concentra- tion was checked occasionally by the pycnometric method. The integrity of the compounds studied at high acidity was checked neutralizing the solution and comparing its absorp- tion and fluorescence spectra with those of the original sample, before acidification. The pK, of the ring (4') ammonium group in the investigated compounds were determined graphically from the spectrophotometric titra- tion curve.

The difference in the dissociation energy of the ammo- nium group in the aromatic ring of Aphe and in peptides and esthers containing Aphe was calculated from Eq. 2 (Kor- tiim, 1970).

A(AG) = - 2.303 RT (PIC,, - pICa,) (2) where the subscripts 1 and 2 refer to the unprotected Aphe and the Aphe derivative, respectively. R is the gas constant and T is the absolute temperature.

The pK,* value of the 4' ammonium groupof Aphe in the excited state was calcuiated from Eq. 3 (Weller, 1961)

0.625 A; T

PK,* = pK, - ___ (3)

where All is the o - o band shift in cm-' of the cationic and neutral forms of Aphe. (AG was determined from the intersection points of absorption and fluorescence spectra (Fig. 2) (Grabowski and Grabowska, 1976). In Eq. 3 the correction for the change of hydration entropy of aromatic amines on their excitation was applied (Rotkiewicz and Grabowski, 1969).

Fluorescence lifetimes. Fluorescence decay curves were measured with an SP-3 nanosecond fluorescence spectro- meter (ORTEC and Applied Photophysics). The excitation light at 295 nm was obtained with a nitrogen-filled (0.5 atm.) flash lamp and a monochromator. The emission was observed through a 405 nm interference filter (Zeiss, E. Germany). The fluorescence lifetimes were computed from decay data by the method of moments (Isenberg er a[ . , 1973).

IR spectra. IR spectra were recorded on a Specord IR 75 (C. Zeiss, E. Germany). Samples were dissolved in metha- nol, placed in KRSJ mounting cells and dried to give a thin film of about 0.05 nm.

RESULTS

Characterization of Aphe as a fluorescent label

Parameters of the fluorescence spectra of Aphe in the unprotected form and of the Aphe residue in A-enk in aqueous solutions at p H 5-7 are given in Table 1. The fluorescence quantum yield of the unprotected Aphe is comparable to those of the tryptophan and tyrosine residues in natural proteins (Cowgill, 1976; Schiller et al. , 1977). Incorporation of Aphe into the peptide chain of A-enk lowers the fluorescence yield by about 50% and reduces prop- ortionally the excited state lifetime. This prop- ortionality suggests that intramolecular fluorescence quenching of Aphe by peptide bonds is principally dynamic. A similar quenching interaction probably occurs in the case of tyrosine, where the effect is

much stronger. The fluorescence quantum yield of tyrosine in peptides is much lower than that of the Aphe residue in A-enk and in other peptides (cf. Cowgill, 1976 and Table 2). Therefore Aphe seems to b e more desirable than tyrosine for structural fluorescence investigations of peptides.

The usefulness of Aphe in this respect depends also on its absorption characteristics. The intensities of the absorption bands, fluorescence quantum yields and the comparison of the spectral band positions of the investigated compounds are given in Table 2 . The absorption spectra of the Aphe derivatives are due to peptide bonds in the wavelength range 200 - 230 nm. Whereas in the range 230 - 300 nm two bands are visible which are characteristic for aniline deriva- tives. In our comparison of the absorption and emission spectra of Aphe and related compounds (Table 2), Aphe is treated as an aniline and phenyla- lanine analog. The summation of the substituent shifts for aniline and phenylalanine (relative to benzene) gives the bands positions identical to those observed for Aphe (Table 2). For esthers such as APGGOEt and Aphe OMe a small but distinct red shift of 'La - 'A band, relative to the corresponding unprotected Aphe band is observed. The fluoresc- ence bands of Aphe OMe and APGGOEt are even more markedly shifted to the red relative to the Aphe emission band.

Solvent effects in absorption spectra of Aphe (Table 3), analogously to the spectra of aniline (Suppan, 1974), depend on proton donating ability of the solvent. In protic solvents the absorption bands of Aphe are blue shifted with increasing dielectric constant. The effect in protic solvents is undoubtedly due to the stabilization of the ground electronic state of Aphe by a hydrogen bond, with the solvent (Suppan, 1974). Fluorescence spectra of Aphe are red shifted with increasing dielectric constant of the solvent, independently of its proton donating ability. This suggests that the excited state is not influenced by the hydrogen bond with the solvent. No linear dependence of band maxima on solvatochromic para- meters was observed.

The spectral effects in aprotic solvents reflect dipolar interactions between chromophores and sol- vent. These effects were used to calculate (by means of Eq. 1) the change of chromophore dipole moment on the excitation (Ap*). Results are given in Table 4. Solvent effects in the electronic spectra of Aphe suggest that spectral properties of this residue in a peptide or membrane hormone receptor may change depending on hydrophobicity or proton donating ability of chromophore microenvironment.

Spectrophotornetric titration

Dependence of the extinction coefficient of Aphe and APGGOEt at 250 nm on p H of the solution is shown in Fig. 1. Absorption spectra of Aphe in a pure acidic form (pH 2 ) and in a conjugate base form (pH 7) are shown in Fig. 2. Similar absorption changes were also obtained with other compounds.

4'-Amino phenylalanine 161

Table 1. Fluorescence properties of unprotected Aphe and of Aphe residue in A-enk

Lifetime Fluorescence of the

quantum excited Compound yield st (ns)

(4) 7

Position of the

excitation spectrum maximum

~ m a x

(nm)

Position of the

Extinction fluorescence coefficient spectrum

(cm'mM-') maximum (285 nm) hrn't,

(nm)

Aphe 0.086* 9.8 Aphe residue 0.04 5.7

in A-enk

285 285t

1360 345 3 4 3

*For determining the fluorescence quantum efficiency of Aphe tryptophan was used ;is a standard.

tThe positions of the corrected excitation spectrum maximum and of emission spectrum maximum for A-enk were determined with less accuracy than for unsubstituted Aphe because of overlapping tyrosine and Aphe spectra in the pentapeptide.

Table 2. Comparison of the absorption and fluorescence spectra of the investigated compounds in water solution

Compound

Absorption Fluorescence

Extinction Quantum h l a x coeff. &la, yield (nm) (cm' mM-') (nm) 4

Aphe - experimental value

Aphe - value calculated from the substituent shifts)* Conjugate acid of Aphe - ApheH+ Aphe OMe

APGGOEt

Anilinet

Phenylalanine

Benzene

284.3 236.0 285.9 235.8

258 213 285.1 238.0 285.7 237.6 281 .0 230 258.0$ 208.5$ 254.0$ 203.0$

1360 345 0.086 8900

170 282 0.01

1360 350 0.014 8900 1360 348 0.067 8900

344 0.024

282$ 0.035$

275.6$

*This value was obtained by the following procedure: the sum of the shifts (AC) for aniline and phenylalanine absorption bands relative to the correspondent bands of benzene in cm- ' was added to the value of band maximum of benzene and the obtained value was converted to nm.

?Data taken from Bridges and Williams (1968). $Data taken from Leroy et al. (1971) or from Jaffe and Orchin (1965). $Our data (phenylalanine, FLUKA and benzene, MERCK, ultrapure were used).

This titration behaviour, is undoubtedly due to protonation of the 4' amino group of Aphe. This implies localization of nitrogen nonbonding electrons near this nucleus and the aromatic ring of Aphe H+ is nearly isoelectronic to that of phenylalanine. The analogy between Aphe H+ and phenylalanine (Jaffe and Orchin, 1965) is used below in the interpretation of our experimental results.

The pK, values of the 4' ammonium group in the ground s ta te of the investigated compounds, obtained from their spectrophotornetric titrations, are listed in Table 5. All these values are close to that of aniline. However pK,'s of Aphe OMe and APGGOEt are lower than the pK, value of aniline, while the value of Aphe is higher. The pK, value of A-enk is less certain because of a tyrosine component

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4'-Amino phenylalanine 163

Table 4. Change of the dipole moment of Aphe on its excitation to the lowest singlet excited state calculated from Eq. 1 (Ak*)

Solvent Dielectric Refractive Ak* constant index (v, - v,)t (D)

(D) (n)

Acetonitrile 37 .3 1.34604$ 3898.9 -_ 9ll Dime thyl-sulphoxide 45.09 1.4187' 3882.6 3.11'

tDifference between maximum of the absorption spectrum (Lh band) and that of

$Data taken from Landold-Bornstein op. cit. 'Data taken from The Merck Index 1960. //This value is close to analogous value for aniline (Mataga, 1963).

the fluorescence spectrum.

Table 5 . Values of pK, of 4' ammonium group and differences in proton dissociation energy for this group, between the investigated compounds and unprotected

Aphe calculated from Eq. 2

Compound PK, 4 A G ) (callmol)

Unprotected Aphe 4.72 APGGOEt 4.25 632.4t Aphe OMe 4.00 968.7t Aphe residue in A-enk 5.02 Aniline* 4.61

*D. D. Perrin, Dissociation constants of Organic Basesin Aq. Sol.; IUPAC, Butterwords London 1965.

tThese values are close to the shifts of 'La band positions, relative to unprotected Aphe band positions (Table 2) which are 829 calimol for APGGOEt and 1000 callmol for Aphe OMe.

in absorption spectrum. Differences in proton dis- sociation energy of 4' ammonium group between the investigated compounds (Table 5) were calculated from pK, values by means of Eq. 2.

Syectropuorimetric titration

It can be seen in Fig. 1 that in Aphe solution at p H 0 - 2.5 only the acidic form Aphe H+ is present. However the solutions in the same p H range show fluorescence spectra with a maximum at 345 nm, identical with those of samples at p H * pK, which contain only the neutral form Aphe. This suggests that in the excited state Aphe H+ undergoes adiaba- tic deprotonation according to Eq. 4 whereby the deprotonated form Aphe is the fluorescent species.

Aphe H+ + H20 Aphe + H30+ (4)

Fluorescence spectra of Aphe at highly acid solutions are shown in Fig. 3. Two fluorescence bands with maxima of 282 nm and at 345 nm are observed in 3670% H2S04 corresponding to -5 < Ho < -2. The short wavelength band (282 nm) is of almost the same energy as phenylalanine emission band (Ae,, = 282 nm) while the long wavelength band (345 nm)

kl

k- l

E E.,,t ..

1.0

0.8

0.6 -

0.4r

-

-

1.0

0.8

0.6 -

-

-

- . - 2 3 4 5 6 ? 8

PH

Figure 1. Spectrophotometric titration of Aphe and APGGOEt . Curve 1 unprotected Aphe. Curve 2

APGGOEt.

Y - 7 0 I 1

---T--- , .. 4 4 0 480

WAVELENGTH, nrn

Figure 2. Normalized absorption and fluorescence spectra of the cation (ApheH+) and of the neutral form (Aphe). Curve 1 cation absorption spectrum (lowest energy band). Curve 2 cation fluorescence spectrum (lowest energy band). Curve 3 neutral form absorption spectrum (lowest energy band). Curve 4 neutral form fluorescence spectrum. Curves

1-2 were multiplied by the factor of 10.

coincides in position with the emission band of the deprotonated Aphe. Therefore, these bands are attributable to the protonated ApheH' and to depro- tonated Aphe species, respectively. Similar cation fluorescence was observed for Aphe OMe and APGGOEt , whereas no short wavelength fluoresc- ence band was observed for A-enk; probably it is hidden under stronger tyrosine emission.

164 ANDRZEJ JANKOWSKI and PIOTR DOBRYSZYCKI

WAVELENGTH, nm

Figure 3. Fluorescence spectra of Aphe at high acidity of the solution. Curve 1 64% H2S04 Ho = -4.95. Curve 2 50% H2S04 H, = -3.38. Curve 3 42% H2S04 Hn = -2.60.

Curve 4 37% H2S04 H, = -2.25.

The results of the spectrofluorometric titration of the unprotected Aphe, Aphe OMe, APGGOEt and of Aphe residue in A-enk are shown in Fig. 4, where the relative fluorescence quantum yields +/I$() are plotted against pH/Ho. $I and $0 are the relative fluorescence quantum yields at a given acidity and at

Spectrofluorometric titration curves for the unpro- tected Aphe and A-enk residue differ appreciably from those of Aphe OMe and APGGOEt (Fig. 4). However the deviation is not related to the kinetics or equilibrium of the reaction given by Eq. 4 but is due to intramolecular quenching of the fluorescence by protonated carboxyl group at pH < 3 - a phe- nomenon analogous to the quenching of tyrosine fluorescence by protonated carboxyl (Cowgill, 1976).

of Aphe residue in A-enk begins to decrease at Ho < -1. This is probably caused by fluorescence quenching through the leucyl carboxyl group in the pentapeptide. The reason why the quenching is less effective than in the case of the unprotected Aphe might be a larger distance between the chromophore and the quenching group.

The fluorescence quantum yield of Aphe OMe and APGGOEt was found to be almost unchanged from pH 7 to Ho 3 - 3 as expressed by I$/& = 1 within this acidity range (Fig. 4 curve 3). At H0 < -3 there is a decrease of the relative fluorescence quantum yield of Aphe OMe and APGGOEt accompanied by a cation fluorescence.

The +‘/+o‘ curve for the cationic form could not be constructed directly from our results as the values of +A for Aphe H+ (fluorescence quantum yield at H(, 4 pK;) is unknown. We assumed $6 equal to the

PH ’ PKa.

fluorescence yield of phenylalanine in H2S04 and 4’16) was expressed as the ratio of fluorescence quantum yield of the sample at 282 nm to that of phenylalanine at the same conditions. In this way the curve 4 in Fig. 4 was obtained.

By means of Eq. 3 pK,* = -5.5 for excited state proton transfer reaction of Aphe was calculated. The spectrophotometric titration curves of +/+o and + I / + ;

for Aphe OMe and APGGOEt intersect at Ho = -4.75. This value is close to the pK,* value of Aphe.

The spectrofluorimetric titration curves of Aphe OMe and APGGOEt are characterized by a sharp transition from pure cation emission (A,,, = 282 nm) to its conjugate base fluorescence (A,,, = 345 nm). These two fluorescence bands are simultaneously observed in the acidity range, -5 < Hn < -2.5. This behaviour has to be compared with the spec- trofluorimetric titration results of some aromatic acids and bases given in the literature (Weller, 1961). In those cases where the equilibrium of the excited state dissociation reaction is not reached during the excited state lifetime, dual fluorescence from an acid and its conjugate base appears over an acidity range of at least 4 pH/Ho units. The intersection point of respective spectrofluorimetric titration curves in such cases is usually far away from pH/Ho = pK,*.

A comparison with the data cited in the literature (Watkins, 1972; Schulman, 1980; Vogt and Schul- man, 1983) clearly demonstrates that in the case of Aphe the equilibrium of excited state proton transfer (Eq. 4) is established during the excited state life- time. It can be concluded that the excited state deprotonation of Aphe Hf is probably so fast that the ’

cation emission of Aphe derivatives would not be observed unless at very high acidity.

Aphe as an acceptor in fluorescence energy transfer experiments

The Forster’s theory (Forster, 1965) of very weak dipole-dipole interaction makes possible the deter- mination of intramolecular distances in peptides. In our previous work (Jankowski et al., 1981) the efficiency of energy transfer fro-m tyrosine to Aphe in

P H / H o

Figure 4. Spectrofluorimetric titration of the investigated compounds. Curve 1 unprotected Aphe-deprotonated form. Curve 2 A-enk deprotonated form of Aphe residue. Curve 3 Aphe OMe 0-0-0 and APGGOEt .-.-. deproton-

ated form. Curve 4 Aphe OMe cationic form.

4’-Amino phenylalanine 165

A-enk was measured and 10.4 A was obtained for the intramolecular distance between the aromatic rings. This result is in agreement with the previous measurements of Schiller er a/ . (1977) carried out on Try4 - Leu’ enkephalin analog as well as with the results of Guyon-Gruaz et al. (1981) and Kuprys- zewska et a/ . (1982) obtained with dansylated en- kephalin analog and an unmodified enkephalin, respectively.

The use of Aphe as an acceptor offers improve- ment in determining intramolecular distances since the bleaching of Aphe due to its protonation enables direct and simple determination of fluorescence quantum yield of the donor (4%). This can be explained by considering the relationship between the rate of energy transfer kT and donor-acceptor distance ( R ) , which is expressed by Eq. 5 (Dale and Eisinger, 1975).

where +: is the donor fluorescence quantum yield in the absence of energy transfer, K is the donor- acceptor orientation factor, T~ is the lifetime of the

donor excited state in the absence of energy transfer, N’ is Avogadro number per cm3 and n is the refractive index of the medium. The overlap integral J A D is given by Eq. 6

where FA and Ah are the normalized fluorescence intensity of the donor and the molar extinction coefficient of the acceptor at wavelength A , respec- tively.

It is evident that protonation of ring (4’) amino group of Aphe causes an almost complete loss of its absorption at h 3 280 nm (Fig. 2), where the fluorescence of most aromatic compounds begins. Thus the protonation results in the disappearance of the overlap integralJAD. From Eq. 5 it is obvious that if J A D = 0 (in the case of protonated Aphe) the probability of energy transfer (kT) vanishes. This is very advantageous for the measurement of donor fluorescence quantum yield in the absence of energy transfer (4:). Obviously energy transfer efficiency has to be determined with deprotonated Aphe. It must be noted that the estimation of 4: is absolutely necessary for intramolecular distances determina- tion.

The change of Aphe absorption spectrum on its protonation may be also utilized for separating the Aphe fluorescence band from that of another emitter in systems containing, besides Aphe, other fluorophores with similar spectral properties (e.g. tryptophan). For this purpose the fluorescence spec- trum of the system at acidic solution (protonated Aphe) should be subtracted from the spectrum recorded at neutral pH (deprotonated Aphe). The difference fluorescence spectrum gives Aphe compo-

nent in the emission spectrum of the system. It can be seen from Fig. 1 that at pH < 3 only the

protonated form Aphe Hf is present in solution. Therefore, the pH value of solution has to be lower than 3 in order to determine & and/or trace the Aphe component in the fluorescence spectrum of a system.

Experiments were performed at pH = 3.3 since this pH yielded almost the same result as at pH 3 (Jankowski et al., 1981).

As can be seen in Fig. 2, the protonation of 4’ amino group of Aphe greatly enhances the excited state energy level of the chromophore. However it was proved in “spectrofluorimetric titration” section that at low acidity (pH 3) at which the determination of 4: is to be performed, the rate of deprotonation reaction (Eq. 4) would be much higher than the rate of energy transfer (Eq. 5 ) . Thus the changes of absorption spectrum of Aphe at pH 3 would not damage 4; determination.

Finally the possibility of energy transfer from Aphe Hf to tyrosine in A-enk was examined. The fluorescence spectrum of A-enk at pH 1.7 is shown in Fig. 5. According to Fig. 1 Aphe residue should be, at this pH value, in the cationic form. The efficiency of energy transfer from Aphe H+ to tyrosine was calculated from the emission of the acceptor. The measurements, repeated several times, resulted al- ways in the transfer efficiency value equal to zero. On the other hand, in unmodified leucyl-enkephalin (Tyr-Gly-Gly-Phe-Leu) which represents an analog of A-enk, energy transfer efficiency from phenylala- nine to tyrosine is found to be very high ( T = 0.9 - see Kupryszewska et al., 1982). Since Aphe Hf is an analog of phenylalanine the observed complete lack of energy transfer from Aphe H+ to tyrosine in A-enk is unexpected. This result indicates that the deprotonation rate of the excited Aphe H+ is much higher, compared to the (very high) rate of energy transfer.

Figure 5 . Fluorescence spectrum of A-enk at pH 1.75 excited at 250 nm. Solid line-registered spectrum. Curve 1

tyrosine contribution. Curve 2 Aphe contribution.

166 ANDRZEJ JANKOWSKI and PIOTR DOBRYSZYCKI

DISCUSSION

Our results (UV, IR) do not suggest the presence of an intramolecular hydrogen bond of the ring (4’) amino group in Aphe (Scheme 1) and its derivatives. The electronic spectra of Aphe can be considered as analogous to those of aniline. This analogy may be helpful in theoretical interpretation of the spectra of Aphe residue in peptides. In the present paper Aphe was proved to be very useful as an energy acceptor in energy transfer experiments in peptides.

Aphe as a fluorescent label gives the possibility of correlation the pK, values of the compounds studied with the absorption band positions. It should be noted that the differences in proton dissociation energy of these compounds [A(AG)-Table 51 are concordant in sign and close in value to the differ- ences in absorption band positions (Table 2; Table 5 note b). This suggests that the substituents in the position 1’ (Scheme 1) in APGGOEt and Aphe OMe increase the extent of electron migration from nit- rogen to the aromatic ring in the ground state. It results in an increase of the acidity in the 4‘ ammo- nium group and concomitant enhancement of the highest occupied ring orbital energy level, which is expressed by the bathochromic shift of the absorp- tion band, relative to unprotected Aphe absorption band.

A favorable property of Aphe as a fluorescent probe is the possibility to use it for further modifica- tion of the labelled peptide by its diazotization and linking with another peptide or protein by an azo bridge. Such an approach was utilized by Szewczuk et af. (1983) in immunochemical studies. For possibili- ties of additional modifications see Eberle (1983).

Efforts to introduce selectively a modified Aphe into a protein molecule are in progress in our laboratory. An interesting phenomenon is the fluorescence of the cation (Aphe H+) with maximum at 282 nm. This property of Aphe H+ is in an apparent contradiction to the general analogy be- tween Aphe and aniline since aniline cation is non fluorescent (Bridges and Williams, 1968). This differ- ence between Aphe and aniline may be due to the collisionai quenching of aniline cation fluorescence by protons (Druzhinin and Uzhinov, 1983) which would be less effective in the case of Aphe because of

NH2 I

Scheme 1. Aphe-structural formula.

the ring protecting effect exerted by the substituent in the position 1’ (Scheme 1). This suggestion is sup- ported by our finding that phenylalanine, an analog of Aphe H+, is fluorescent in 64% H2S04 while benzene (analog of aniline cation) is non fluorescent at exactly the same conditions.

Acknowledgements-We are grateful to Alexander v. Hum- boldt Stiftung, Bonn, W. Germany for making accessible the spectrofluorimeter. We are indebted to Professor Dr. I . Z. Siemion and to Professor Dr. M. Kochman for fruitful discussions and to Mr P Stefanowicz for supplying us with a sample of APGGOEt. This work was partly supported by Polish Ministry of Science and Higher Education Grant MR. 1.9 and by Technical University of Wroclaw Grant and Polish Academy of Science Grant 3.13.4.4.1.

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