Primary photoprocesses in retinol

Volume 20, number 3

CHEhfICAL PHYSICS LETTERS

PRIMARY P~~T~PR~CESSES Tchiya ROSENFELD, Aron ALCHALAL Department

of Physical Ciremistry,

1 June 1973

IN RETINk and Michael OTTOLENGHI

The Hebrew Utrirvrdty, Jerusalem, Israel

Received 26 March 1973

Pulsed laser photolysis techniques, with nanosecond time-resolution, are applied to solutions of retinal ftitamin A) and retinyl acetate, under varying conditions of solvent polarity and oxygen content. Tbc observed absorkmce and conductivity changes are identified as due to: (a) the excited fluorescent state, hax = 435 nm; (b) the towest escited tripiet state, h,, = 40.5 nm; (c) the retinylic cation (kmas = 590 rim), formed via: ROH k RC f OH-. The re-

sults constitute the first e~perimentai evidence for ionic photodi~sociat~on and for spontaneous, as weti as osygeninduced, intcrsystem crossing in retinol. Both processes may provide routes for isomerizstion, being thus relevant to the \lisunl process.

1. Introduction The understanding of the primary steps in the visual process is closely associated with radiationless deactivation paths in excited pigments, such as rhodopsin. It is also recognized that the photache.micaI behaviour of retinal (the polyene chromophore of rhodopsin) and its derivatives, such~as the corresponding dcohoi (retiool or vitamin A) as well as the protonated and unprotonated Schiff s bases, may in some degree ret? ect the photochemistry of rhodopsin [ 13. In the case of retinal, attention has been mainly paid to cis-tram photoisomerization processes [2]. Indirect information on non-radiative losses has also been obtained from fluorescence studies [3,4]. However,‘no direct experimental evidence appears to be available concerning radiationless deactivation paths in ietinol which involve intersystem crossing (isc) and ionization. The possi&z relevance of both processes to photoisomerization and, more generally, to the early stages of vision, has been repeatedly considered 11, 5, S] . As far as isc is concerned, tlash photolysis experiments in deaerated solutions [7,8] have actnally led to the detection of the lowest triplet state of retinal, formed with a q-danturn yield of 0.6 [9]. However, dthough populated using photosensitization [lo) or pulse radiolysis [II) methads, the triplet statr: of, vitamin A (or of a ?rot~nated Scfliff’s base of retinal)

have not been detected by applying direct flash excitation, Abr~~son and coworkers attributed &se observations to the inefficiency of intersystem crossing in the latter cases, due to the lack of a suitably situated m-r* state similar to that which, according to them, is responsibie for a rapid r(n;i*) + 3(~z”) transition in retinal.- As long as rhodopsin is considered to be a protonated Schiffs base [I2], these assumptions appeared to rule out the participation of triplet states in the early stages of vision [ l] . In the present work we have examinecf these considerations by submitting solutions of retinol and retinyl-acetate to nanosecond pulsed-Iaser excitation. The study ieads for the first time to the observatiun of both intersystem crossing and ionic photodissociation as primary processes in the photochemistry of these molecules.

2. Experimental The bask-pho~ochemi~~ technique using the 337.1 nm excitation line of an Avco-Everett pulsed (10 nsec, 0.5 mJ) nitrogen laser has been previously described 1133. Steady state tluorescence experiments were carried out using a Turner model 2 10 fluorimeter. AU-tram retinol obtained from Sigma Che,mical Co.

or from ~ist~a~o~ Products Industries wak used wit.b .’

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a

Fig. 2. Characteristic

oscillograms and kinetic data showing (B-C) and photosensitized (D-F) generation of the triplet state of retinol in )Ihexnne solutions. (A) 5 X IO3 hi retinal, N2 saturated, >. = 405 nm. (B) As A, air saturated. (C) As A, 62 saturated. (D) 5 X 1O-s 51 retinal, 5 X 10m2 hi TMPD and eO.1 M biphenyl, N2 saturated, h = 370 nm. (E) As D, h = 405 nm. (F) Reciprocal half life of biphenyl triplet as function of rerinol concentration. the

Fig. 1. Excited singlet-singlet absorbance changes due to the fluorescent state of retinol (5 X 10W5 hi in deaerated ?Ihexane). (a) Characteristic oscillogrnms recorded at 435 nm. Upper trace: fluorescence only (without monitoring beam). Lower trace: absorption + fluorescence (with monitoring beam). tb) Spectrum drawn from absorbance changes recorded (after appropriate correction for the contribution of fluorescence) at the peak of absorbance change (=I0 nsec after triggering).

out further purification. Eastman Kodak biphenyl and N,N,N’,N’ tetramethyl-p-phenylenediamine (TMPD) were zone refined. Fluka spectrograde solvents (methanol, acetonitrile and n-hexane) were used w’ithout fur*&er purification. Solutions were deaerated or oxygen saturated by bubbling correspondingly nitrogen and oxygen. Experiments were carried out with freshly prepared solutions under deep red light.

3. Results and discussion 3.1. General kzser-photolysis patterns Upon submitting deaerated solutions of dl-tram 292

spontaneous (A), oxygen-induced

retinol or retinyl-acetate to the 337.1 nm N, laser excitation, transient absorbance changes are observed which can be rationalized in terms of three species: (a) a short-lived transient characterized by an absorbance maximum around 435 nm and by a decay which closely follows the fluorescence profde (7 < 10 nsec) (fig. 1j; (b) a species absorbing very weakly around 405 nm, decaying in the microsecond range (figs. 2A, 3A); (c) a third species, which is only observed in polar solvents such as methanol or acetonitrile, absorbing around 590 nm and decaying in the microsecond range (fig. 4). Polar solutions of retinol and retinyl-acetate were also submitted to laser-excitation experiments in which conductivity changes were recorded. Fig. 4 shows a characteristic photocurrent trace for all-tram retinyl-acetate in acetonitrile. Similar, though weaker, photo-conductivity signals are also observed in retinol solutions.

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I June 1973

AIR SATURATED

DEAER4TED

Fig. 3. Transient triplet spectra in the laser photolysis of retinol solutions in cyc!ohesane. (A) 5 X 1O”j hf retinal in aerated and deaerated solutions. Data recorded 50 nsec after tiring the laser pulse. (B) 5 X 10 -5 hi retinal, 5 X lo-* hf TMPD and ==O.i Xf biphenyl, 200 nsec and 1.5 ~sec after the pulse.

3.2. T?le 40.5 nm transient As demonstrated in fig. 2 the yield of Ihe 405 nm transient absorption increases markedly in the presence of dissolved oxygen. A similar enhancement was

also observed when bromobenzene was added to deaerated solutions. Fig. 2 shows that the rate of the transient decay is affected by dissolved oxygen? the second order quenching rate constant being ~10~

M-l

set-I.

We have also been able to produce the 405 nm species via energy transfer to retinol from the triplet state of biphenyl. This is demonstrated by the characteristic oscillograms D, E in fig. 2, recorded in a cyclohexane solution of 5 X low2 TMPD, 0.1 biphenyl (B) and 5 X 10e5 M retinol (ROH). In this system more than 8G% of the 337.1 nm laser line are absorbed by TMPD, leading to the fluorescent state of the molecule which undergoes subsequent quenching by biphenyl, yielding a charge-transfer fluorescent exciplex [14] : ‘TMPD* + B + ‘(TMPD - B)* .

(1)

Intersystem crossing in the thermalized complex leads to the triplet state of biphenyl [ 141 : ‘(TMPD - B)* + TMPD + 3B* .

G9

(3B’ could not be populated by direct excitation of B since the 337.1 run laser !ine is not absorbed by bipheny!.) The addition of retinol to the TMPD-biphenyl system shortens the iifetime of the 370 MI absorbance of 3B*. A characteristic osciilogram showing the decay cf 3B* in the presence of retinol and a plot of its half life as function of the retinol concentration (leading to a quenching rate constant of ~3 X IO9 M-l see-‘) are shown in figs. 2D, F. A comparison between oscillograms D and E in fig. 2 indicates that in the presence of retinol the 3Be decay at 370 nm B accompanied by a matching growing-in process around 405 nmf .(The very fast initial drop in absorbance at 40.5 nm, seen in fig. 2E, is due to the ~50 nsec decay of the exciplex [ 141.) Fig. 3B shows the complete spectra recorded 200 nsec and L.5 wee after triggering the laser. The initial spectrum consists mainly of the characteristic 370 nm band of the biphenyl triplet [ 151. The curve recorded after I .5 psec shows the identity between the spectrum of the growing-in species and that of the 405 nm transient obtained by direct excitation of retinol in the presence A detailed kinetic analysis quantitatively comparing the decay of 3B* with the growing-in of the retinot tripIet (ROH’), was not carried out due to the comptickons associated with the superimposed decay of 3ROH* (fig. 2).

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,,I/a 10

30

20

F” , 0

F

540

620

580

X, nm

Fig. 4. Transient absorbance and conductivity

changes in polar aerated solutions of retinyl+cetate. (a) Decay of photoconductivity in it IO 4 hl solution in acetonitrile. E!ectrode voltage: 90 V. (b) Absorbance decay in a 5 X tOe5 M solution in methanol. The Ii&t to dark deflection is 120 mV. (cl Points and dotted lines are absorbance changes recorded IO0 nsec after the pulse in a 5 X 10e5 M solution in methanol. Solid lint is spectrum of retinylic cation recorded by Blntz and Pippert 1221 in n-butanoi. The absorbance maximum reported by them in ~tcl~aRo~is at 589 nm.

or absence of dissolved oxygen. This estabIishes the trip&t energy transfer process: 3B* + ROH + B + 3ROH* ,

I,‘. ‘, ‘,

-

.,

50

’

Fig. 5. Plot of oxygen quenching data for retinol in n-hexane according to cq. (4). D=,lDF was estimated from the correspondin:: initiaf triplet absorbance changes at 405 nm. The relative fluorescence parameter was estimated as follows: (a) 0 From areas under the excited singlet-singlet absorbance profile (F”/F-@/Sj. (b) a From maxima of fluorescence sinds measured at 495 nm on the laser apparatus (Fe/F= Fi/F,j. (c) l From relative fluorescence intensities measured at 470 nm on the steady-state fluorimeter (Fe/F = FfJF,).

sorbance relative to deaerated solutions, are consistent with the triplet assignment of this species. Assuming f16], as has been recently directly verified for a series of aromatic molecules in a non-polar solution [ 171 I that the oxygen quenching process exclusively involves intersystem crossing, producing one triplet molecule for each singlet quenched, the following scheme applies: ROH --f ‘ROH* ,

(31

identifying the 405 nm species formed by direct excitation of retina; as the triplet state of the molecule_ The weak 4%nm absorbance present in the photosensitization experiment after ~200 nsec should partially be attributed to a small amount of 3ROH* fo&ed by direct excitation of ROH, and, partialfy, to the. contribution of the early stages of reaction (3). The results obtained in the presence of dissolved .-oxygen, showing an e&&cement of the 40.5 nm ab,294 : ‘.. _.

D’

40

‘ROH’ + ROH + hv , ‘ROH+ -+ 3ROH* , IROH* f o2 + 3~~~* f 0, , leading co the expression [13] :

: :

:

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CHEMICAL PHYSICS LETTERS

where F” and F are, respectively, the fluorescence intensities in the absence and in the presence of 02.0: and D, are the corresponding absorbance changes due to the triplet state and 4 is the triplet yield in the absence of a quencher. A plot of F”IF versus (Fo/F)(D,/D$) - 1 (fig. 5) is found to be linear with an intercept of ~1, confirming the above mechanism and yielding $I; = (6 f 3) X lo-* for the yield of intersystem crossing in retinol. The substantial error in the evaluation of & is due to the difficulty associated with the exact determination of the triplet absorbance, D!, in deaerated solutions. The above (dynamic) quenching mechanism is consistent with a Stern-Volmer analysis using 7 = 4.4 nsec for the fluorescence lifetime of retinol in deaerated IIhexane [4]+ and X-IROH*+~~ = 2 X lOlo M-l set-t. Static quenching was ruled out by our failure to observe any oxygen effects on either absorption or emission spectra of retinol and retinyl-acetate, as well as by the independence of the parameter +/rF (where + is the fluorescence yield and TF is the measured lifetime) on the O2 concentration. Thus, the same value@F/rF =6X 10P12 set-l-i- was obtained in deaerated, aerated and oxygen saturated solutions, implying that the drop in the fluorescence yield, QF = kF TF (where kF is the rate of fluorescence emission), is quantitatively accounted for by a dynamic quenching of lROH* by 0,. 3.3. The 435 iIf

transient

As pointed out previously, the laser-induced change in absorbance due to the 435 nm transient follows a curve which is close to that of the fluorescence profile. The lifetime of this absorbing species is smaller than the duration of our laser pulse (~10 nsec), and it is difficult to carry out a detailed deconvolution analysis determining the absorbance lifetime and subsequently comparing it with that of the fluorescence. However, strong evidence supporting the identification of the 435 nm band with an excited singlet-singlet transition of vitamin A in its fluorescent state, can be derived from both oxygen and solvent effects. Fig. 5 shows that t’le relative lifetime of the 435 nm transient which is proportional to the area under the correspondt Unpublished data-in this laboratory, using the value $F = 0.02 in aerated n-hexane solution [ 31.

1 June 1973

ing absorption versus time curves, varies with the oxygen concentration in the same way as the relative fluorescence lifetime. In other words, within the limits of our experimental accuracy we observe: L$/Fs = Foe/Fe = so/S, where the subscripts s and Q refer to

fluorescence intensities determined correspondingly by using the steady state fluorimeter and by measuring the areas under the ,emission profile in the Iaser apparatus. .@ and S are the areas under the absorbance profile at 435 run where the contributions of the 405 and 590 nm species are negligible, measured correspondingly in deaerated and in aerated (or oxygen saturated) systems. We have also observed a similar solvent effect on Fs and S, both parameters decreasing by 30 k 10% when going from It-hexme to methanol. All these data support the identificatioi of the 435 run species with the fluorescent state of retinol or retinyl-acetate. 3.4. The 590 nw2 transient The 590 MI species is observed only in polar so!ntions of retinol and retinyl-acetate which are aiso found to be photoconductive. This suggests the identification of this transient with a photoionization product. Since the yieId of the absorbing species in ncetonitrile (where trasient photocurrents were recorded) is considerably smaller than in methanol which is unsuitable for photoconductivity experiments, we have not been able to carry out a quantitative comparison between the decay of the photocurrent and that of the 590 nm absorbance. Such an analysis is further complicated by the highiy focused, nonhomogeneous, geometry of our laser and monitoring light beams [ 131. However, a qualitative comparison (fig. 4) indicates that for retinyi-acetate the two decays take place in the same time range. Et therefore appears that the 590 nm transient should sctualIy be identified with a radical ion, participating in the charge recombination process. The possibility that the 590 nm species is an ion formed via an electron photoejection mechanismi (ROH* -+ ROH+ + e-) is readily ruled out by our failure to observe the characteristic 700 nm absorption of the solvated electron in methanol. Instead the spectral identity shown in fig. 4 very strongly suggests $ For reviews see refs. [ 19, 201. 295

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Volume 20, number 3

1 June 1973

I

Ia‘

I.

Retinol: Retioyl

R z H

-acetate:

REO*COCHj

Ib

.

RO-

I

Scheme 1

the identification of our 590 nm flash transient with ,the retinylic cation (Ia,b) prepared by Blatz and Pippert [21] by placing retinyl-acetate or retinol in a highly acid environment. This establishes the primary photoprocess shown in scheme 1.

4. Conc!tisions The establishment of intersystem crossing and ionic photodissocia$ion as primary events in the photolysis of retinol calls for the investigation of the same phenomena in molecules (such as Schiff’s bases of retinal) which are closer in structure to the rhodopsin chromophore. The enhancement of isc by molecular oxygen, which is knost probably associated with chargetransfer (CT) interactions [17,22], suggests routes to the triplet state of visual pigments involving CT interactions of the chormophores with 0, as well as with electron acceptor (or donor) sites on the protein moiety of the visual pigment. Special attention should now be devoted to the possible involvement of intersystem crossing and ionic photodissociation in the ’ mechanism of the photoisomerization reaction which is known to be associated with the primary step in the visual cycle.

.“,Sxences [ 1) E.W. Abrahamson

and S.E. Ostroy, Progr. Biophys. Mol. Biol. 17 (1967) 179; E.W. Abrahamson and S.M. Japar, in: Handbook of se&o* physiology, Yol. II/l, Photochemistry of vision, ed. H.J.4. DartnaIl (Springer..Berlin, 1972).

296 ‘.

R.H. Hubbard, J. Am. Chem. Sot. 78 (1956) 4662.

;:; k.3. Thomson, J. Chem. Phys. 51 (1969) 4106. 141 J.P. Dalle and B. Rosenberg, Photochem. Photobiol. 12 (1970) 151. [51 F..S. Becker, K. Inuzuka, J. King and D.E. Balke, J. Am. Chem. Sot. 93 (1971) 43; J.R. Weisenfeld and E.W. Abrahamson, Photochem. Photobiol. 8 (1968) 487. I61 B. Rosenberg, Advan. Radiation Biol. 2 (1966) 193; F.J. Grady and D.C. Borg, Biochemistry 7 (1968) 675; CM. Lany, J. Harbous and A.V. Guuo, J. Phys. Chem. 75 (1971) 2861. 171 E. Abrahamson, R. Adamsand V. Wulff, J. Phys. Chem. 63 (1959) 44; W’.Dawson and E.W. Abrahamson, J. Phys. Chem. 66 (1962) 2542. [81 K.H. Grellmann, R. Memming and R. Livingston, J. Am. Chem. Sot. 84 (1962) 546. t91 R. Bensasson, E.J. Land and T.G. Truscott, Photochem. Photobiol. 17 (1973) 53. (101 A. Sykes and T.G. Truscott, Chem. Commun. (1969) 929;Trans. Faraday Sot. 67 (1971) 679. 111; T.G. Truscotr, E.J. Land and A. Sykes, Photochem. Photobiol. 17 (1973) 43. 1121 R.A. Morton and G. Pitt, Biochem. J. 59 (1955) 128. t131 C.R. Goldschmidt, M. Ottolenghi and G. Stein, Israel J. Chem. 8 (1970) 29; hl. Tamir and M. Ottolenghi, Chem. Phys. Letters 6 (I 970) 369. 1141 A. Alchalnl, hf. Tamir and M. Ottolenghi, J. Phys. Chem. 76 (1972) 2229; N. Orbach and M. Ottolenghi, to be published. 1151 G. Porter and M.W. Windsor, Proc. Roy. Sot. A245 (1958) 238. iI61 B. Stevens and B.E. Algar, Chem. Phys. Letters 1 (1967) SG, 219; C.S. Parmenter and J.D. Rau, J. Chem. Phys. 51 (1969) 2242. [I71 R. Potashnik, C.R. Goldschmidt and M. OttoIenghi, Qlem. Phys. Letters 9 (i971) 424.

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[l8f T. Medinger and F. Wkinson,

CHEMICAL PHYSICS LETTERS’

Trans, Faraday Sot. 61

(1965) 620.

[ 191 G. Stein, Actions Chim. Bioi. Radiations 13 (1969) 119. [ZO] R. Lesclaux and J. Joussot-Dubien, in: Organic molecular photophysics, Vol. I, ed. J. Birks (Wley, New York), to be pubLished.

f21] P.E. Bfatz and D.L. Pippert, J. Am. Chem. Sot. 90 (1968) 1296;Chem. Commun. (1968) 176;

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R.S. Fager, P. Sejnowski ar.d: E.W. Abrahamson, Biophys. Res. Cormnun. 47 (2972) 1244.

Biochcm.

1221 H. Tsubomura and R.J. Mull&en, J. km. Chem. Sot. 82 (1960)

5966;

H. Linschitz and L. Pekkarinen, J. Am. Chem. Sot. 82 (1960) 2411; T. Brewer, J. Am. Chem. Sac. 93 (1971) 775.

29;