SPIN RIASONANC~E STUDY OF S~ROTONIAT-AEON INTERACTIONS I~STt'i'~j~~~ FOR MITSC!I.E RESEARCK, MARINE B11~LOGIf~.kL LABORATORY, WOOfH KOLE, IMASSACI-IUSITTS Commztnicakd Au~usl 15, 1060 A variety of indoles form complexes with riboflavin. L. 2 These complexes have been interpreted as ones involving electron transfer, the indoles acting as an elec- tron donor and the riboflavin as an acceptor. Theoretical studies3 have shown that the positions of the energy levels of these molecules are consistent with this interpretation. The indoles have moderately hrgh occupied energy levels, thus making them fairly good donors, while riboflavin has a low unoccupied energy level, thus making it a good electron accept,or. This paper will report a study of serotonin and flavin mononucleotide (I'Mi!!) by the method of electron spin resonance (ESR). Tryptophan and FMN were also studied, but, for reasons stated below, not as extensively. To help identify the spectra obtained, t,he semiyuinone of IGCi at acid pII was prepared by reduc- tion with zinc, by reduction with dithionite and photSoreduction. Tf an electron donor complexes with an cIectron acceptor, the dorror giving an electron to the acceptor, the complex will not necessarily yield a signal in SIZ. In such a complex the donated electBron will not necessarily be completely l~n~oupled from its partner and the two electrons will probably form a weak covalent bond 1308 BIOCHEMIST&Y: ISENUERQ ET AL. PROC. 3. A. 8. and hence the complex will yield no signal. furthermore, even if the electrons were completely uncoupled, two free radicals in such close proximity would broaden each other's signal to a considerable extent by dipole-dipole broadening,", 5 and the resultant signal might be so broad as to completely avoid detection. Conse- quently, the technique of ESIZ, at lea,& undei the conditions reported here, cannot examine the complex direct.ly. It does shed light on the complex, however, since, as will be shown, it indicat'es that, at sufficiently acid pH the complex may dissociate into two free radicals. Ordinary charge transfer complexes, of the type extensively studied by ~~ulliken and his school, cannot do this. Consequently, it appears that, the complexes of indoles and FMN must' be classified different'ly. ,4s suggested previously," the complex appears to bc one ill which the indole gives a ~`~~ornplet,e" clccl:ron to riboflavin,+ resulting in an ionic structure. It might be appropriate to call the ordinary charge transfer complexes cases of "weak charge Iransfer," re- serving the name "strong charge transfer" for complex resulting in an ionic st,ruc- ture. Miller and Wynne-Jones," and Rijl, Kainer, and Rose-Innes,' have examined complexes that also appear t,o be cases of strong charge transfer. Techn.ique.---The ESR apparatus was a Varian 4500~Spcctrometcr', equipped with a 1%inch magnet and a Varian 4560 100 kc modulation unit,. Many features of t,he hypcrfine str~~~t,ure, such as s.yn~metry prop~rt,ies~ arc more cvidellt when the second derivative of the absorption is obtained rather than the first. To achieve this, a dual modulation m&hod, patterned after that of Smaller and Yasai- tes,* was adapted for use with Varian equpmen t,. One side of the 100 kc signal wa.s fed to the Varian 127011, low frequency phase detector. The modulation of both sweeps, but, more especially, t,hat of the low fre~uell~y sweep, wa.s kept very low so that essentially Aecond derivative spectra were obtained. Solutions were cxamilled in flat quartz cells of internal dimcnsious 0.1 mm X 7 mm X 52 mm. The cells were oriented in the microwave c:tvit,y so as lo place the liquid in a mmimum c,lcct,ric field. The FMN was Nu~rition~~l ~io~h~Ini~a1 Corporati~)~~`s ril)~f~~~~~ill 5' ~)hosph~~t,~? Sodium. The serotonin was the California Corporation for Biochemrcal Research Company's serotonin-crentine H&3, comp1ex.S and the tryptophan was the f-l. 34. Chemical Company's J,-tryptophan. The acid used was IfCl double distilled in glass. I'or spectra run at acid pM identical results were obtained over a wide range of acid eoncentratio~~. Thus, while the rcsufts reported below were obtained mainly with undiluted const,ant boiling point HCl, solulions made using a few per cent, HCl gave ident,ical spectra. `I'hc red acid serni~ui~~~)l~~ of FM&' QTLS prepared by dropping zinc inf,o an acid FMN solut8ion and waiting until, the color of the solution became dark red. The solution was then separat,ed from t,he zinc and poured into t,he flat, ccfl. Reduc- tion by dithionite instead of zinc, as well as photoreduction, gave identical results. Phot,oreduct,ion was achieved by t,he method of Commoner and J,ippincott,." The solution was deox~7gei~at,cd and placed for 5 hr at, about 10 inches from a I OO-wal;t desk lamp. Photoreduction at neut,ral pN yielded a different' spectrum from t,he photoreduction at pH < 1. &s&s an.d ~`)iscussion.-lIl'igurc 1 shows Ihc first, derivative of Iho absorption for the red riboflavin fret radical, while I'igurc 2 shows the secorrd derivative. It'igure 2 shows mirror image symmetry with respect6 to the lnidpoillt of the VOL. 46, 1960 HIOCHEMISl'R Y: ISENBERG ET' AL. 1309 1'~;. I. ---First derivative ESR spectrum of 10e2 I\?' FMN in acid solution reduced by zinc. Marker indicates one gauss. Arrow indicates free electron resonance position. 1 H FIG. Z.-Second derivative ESR spectrum of IOW M FMN in acid solution reduced by zinc. Marker indicates one gauss. Arrow indicates free electron resonance position. absorption. This is what is to be expected from a single free radical species. For two free radicals to show such marked symmetry, their g values must coincide. Since this is most unlikely, it seems reasonable to assume that Figures 1 and 2 are the spectra of one species of free radical and that this free radical is the riboflavin semiquinone at pH < 1 .i", l1 Figure 1 demonstrates that the hyperfine structure shows considerable overlap. Attempts to obtain a better resolution of these lines, either by lowering the sweep modulations or by lowering the concentration of FMN, met with no success. At small modulation amplitude the lines do appear to be made up of narrower lines but the sensitivity of the instrument becomes poor under these conditions so that a complete resolution of the spectrum is lacking. Actually, a result such as this is not unexpected. In a molecule such as FMN the spin density will be large at a few places on the molecule. This will give rise to the spectrum shown. The re- maining atoms will have a small but finite spin density. The nitrogen and hydro- gen atoms not giving rise to the large splitting will broaden the lines causing t'hem to overlap. The overlap thus occurs because of unresolved hyperfine structure. Serotonin in acid gave no ESR signal and neit.her.did FMN. Serotonin and FMN, taken together, did yield a signal. Figure 3 shows a second derivative spectrum of serotonin and E'MN. It will be seen that this spectrum is similar but not, identical to the riboflavin semiquinone spectrum. It is markedly asymmetrical, indicating that there are at least two free radicals present. From the similarity to the riboflavin spectrum it may be concluded that one of these is riboflavin. The other may be serotonin. How- ever, since the serotonin free radical spectrum is not known, it may also be a sec- ondary free radical rather than the serotonin free radical itself. The data are there- fore consistent with the following scheme. (1) 1310 l'ROC. N. A. S. Fra. 3.--Second derivative ESR spcctrl~m of s(~rotor~iIl and FMN in acid. Sol~lti~~n was 0.16 114 with respect to FMN and 0.59 M with respect to serotonin. Marker indicates one gauss. Arrow indicates free electron resonance position. PI may not exist. It is included for the reasons just mentioned. Pz and 1':s may be dimers or other products of the free radicals. The symbol S+It- is to be inter-, preted as indicating that the complex is primarily ionic and is not meant to indi- cate that the total charge on the complex is zero. The complex may pick up pro- tons from the solution and hence have a net positive charge. The above scheme may be supported by the following cxpcriment: A solution of 10- 2 IV FMS in acid was placed in a quartz capillary and tested in the spin resonance apparatus. Yo signai was obtained. A small amount of dry serotonin was added to the sample and stirred with a fine platinum wire. This yielded a signal. Succcssivc smail irlcrcments were added and the signal bccamu larger. Finally, however, the signal saturated and further addition of serotonin gave no further change in signal in spite of the fact that the additional scrotonin went int+ solution. At this point, the amount of signal corresponded to one free radical per 250 FMN molecules. The interpretation may be made that the serotonin drove all of the ribofiavin into the complexed form. However, the amount, of free radical present is dctcrmined by the dissociation constant kz and t,his dissociatior~, and hence the amount of free radical in the expt~riln(~l3t just inc~ltioll~~d, is not under the control of the investigator. Tryptophan and Ia'MN in acid also yieIded a signal in ESR. However, Dhe signal was much weaker than with serotonin, so much weaker that t,he hyperfine structure could not be well resolved. This finding is consistent with the obser- vation' that serotonirl is a stronger electron donor than trypt,ophan. The existence of strong charge transfer complexes permits a unified t,heoreti~al framework in the scheme of charge t)ransfer. At the one extreme, for weak donors and/or acceptors, there exist charge transfer complexes of the type bellzene-iodine in which the ground state is overwhelmingly nomonic. For example, it is esti- mated that only about 3 per cent of an electronic charge is transferred from benzene to iodine.12 At the other extreme are t-he well-kno~vn cases of serniqui~lolle forma- tion in which an electron is t)ransferred from the reducing agent to the oxidizing agent, no stable complex being formed. As one considers donors successively stronger than benzene, one passes from weak charge transfer to cases of stronger charge transfer, finally arriving at a predominantly ionic structure. Whether this ionic structure dissociates into free radicals or not depends on whether the free energy of the free radicals, as such, is lower tha.rt that of the co~-nplcx. This, in turn depends, in part, on the nature of the medium, dissociaCol1 being favored by ionizing media such as water. ?;o ESH, signal has been observed when serotonin and Il'MX were mixed at, neutral pH. This is evidence that the complex does not, dissociate into free radi- cals at neutral PH. At acid pH the complex does dissociate, pro~)ably because the riboflavin free radical is more stable in a protonated condition than in an unprotonated state. It may also be, however, that in the complex, at acid pH, the F-MN picks up protons from the medium and becomes a somewhat better acceptor. There may be, therefore, a somewhat stronger charge transfer complex in strong acid. In spite of the power of ESR techniques, a point of caution should be noted. Consider a solution of serotonin and FMN in acid. This is brown at room tem- perature. If the solution is frozen it becomes black, indicating, evidently, a greater degree of charge transfer complexing in the frozen state. Tn spite of this, such a frozen sample, when tested, yielded no ESR signal. It should not be con- cluded from this, however, that no free radical is present. Even if a large number of unpaired electrons were present, ESR techniques might, not detect them. This may easily be shown also by freezing a 1(F3 A4 solution of MnClz. While at room temperature a characteristic &line manganese spectrum is obtained, in the frozen state this signal disappears. One sees, therefore, that electron spin resonance, while a powerful tool, cannot always by itself be relied upon for determining the charge transfer state of a given system. ~~~~u~~.-Evell though the ~chnique of electron spin resonance does not measure a property of the complex directly, it does shed light on the nature of the complex. It seems most unlikely that a weak charge transfer complex could dis- sociate into free radicals. In the case of strong charge transfer, however, under proper env~ronmen~l conditions, such dissociation seems reasonable. Spin reso- nance techniques yield two overIapping spectra, one of which is that of the ribo- flavin free radical. This is evidence that the serotonin-riboflavin complex is of the strong charge transfer or predominantly ionic type and is consistent with data previously obtained using optical absorption methods. The authors wish t,o thank Dr. Bernard Smaller for advice and suggestions in establishing a dual modulation spin resonance system. They also thank Dr. Richard Bersohn for several fruitful discussions concerning the work presented here. * This research was supported by a grant from The Commonwealth Fund, Grant No. H- 204!!( C3) of the National Institutes of Health, a grant from the National Science Foundation, and Grant No. 1 from the Quartermaster Research & Engineering Command, U.S. Army (Natick). 7 The term "complete" is used to indicate that there is a transfer of one, or almost one, elec- tronic charge from indole to riboflavin. $ The authors would like to thank Vism,zra Terapeutici, who supplied part of t,he serotonin used. 1 Isenberg, I,, nnd A. Szent-GyGrgyi, these PROCEEDINGS, 44, 857 (1958). 2 Ibid., 45,1221 (1959). 3 Pullman, B., and A. Pullman, these PROCEEDINGS, 44,1197 (1958). * Van Vleck, J. H., Phys. Rev., 74, 1168 (1948). 0 Weissman, 8. I., J. Chem. Phys., 29,1189 (1958). 6 Miller, R. E., and W. F. I<. Wynne-Jones, J. Chemical Sot., 2375 { 1959). 7 Bijl, U., N. Kainer, and A, C. Rose-Innes, J. Chem. Phys., 30, 765 (1959). 8 Smaller, B., and E. L. Yasaitis, Rev. Sci. In&., 24, 991 (1953). 0 Commoner, B., and B. Lippincott, these PROCEEDINGS, 44, 1110 (1958). 10 Michaelis, L., M. I'. Schubert, and C. V. Smythe, J. Biol. Chm.., 116, 587 (1936). If f-E. Beinert, J. Bm. Chem. Sot., 78, 532:3 (1956). `2 R. S. Mulliken, J. Am. Chem. Sot., `74,811 (1952).