STUDIES ON THE EXCITED STATES OF PROTEINS* By Ricuarp H. Stersiet anp Ausert Szent-Groreyrit INSTITUTE FOR MUSCLE RESEARCH, MARINE BIOLOGICAL LABORATORY, WOODS HOLE, MASSACHUSETTS Communicated March 80, 1958 INTRODUCTION If a bullock lens is laminated by near UV, it emits a brilliant blue light, which is due to its proteins.! This is unexpected because two of the three aromatic amino acids—phenylalanine and tyrosine—have no visible fluorescence, and tryptophane has but a very weak one. the maxima being at 284, 302.5, and 348 mu for phenylalanine, tyrosine, and tryptophane, respectively. These emissions represent transitions from the low- est excited singlet state to the singlet ground state.® Figure 2 gives the phosphorescent spectra for the three amino acids. We have presented data? which led us to interpret. the low-temperature emission for tyrosine and trypto- phane as a transition from the low- est excited triplet state to the singlet ground state. Though the phenyl- alanine emission decay was too rapid for us to make kinetic studies with the equipment we had avail- y a 3 an T Emission, relative intenalt A. L.. 4 i 600 500 400 300 280 dA tn my Fria. 3.—Emission spectrum of intact bullock lens, (a) excited in the 340-my region; (6) excited in the 280-my region. In all figures the wave line marks transmitted exciting light. able, we have interpreted the long- wave-length shift with its close cor- respondence to the tyrosine emission maximum as evidence that this emis- sion isalso triplet-singlet in character. PROTEINS Bullock Lens.—-At room temperature, excitation in the general region of the 280-mu protein absorption band gave rise to an emission with a maximum at 340 my which we consider to be fluorescence originating from tryptophane residues in the protein. Shifting the exciting light to longer wave lengths elicited an intense emission with the same blue color as the phosphorescence we had observed from the same prepara- tion at low temperature (77° K.). Curve 6 in Fig. 3 gives the emission spectra of the bullock lens excited within the protein 280-myz absorption band and by near UV at 77° K. (liquid N,). The 330-my emission band had no delayed emission observable visually on our not too fast in- struments. The 450 mu band, in contrast, exhibited a considerable afterglow. When we first observed the intense blue emission elicited by excitation in the 340-my region, we suspected that we might be inducing a direct singlet-triplet transi- Vou, 44, 1958 BIOCHEMISTRY: STEELE AND SZENT-GYORGYI 543 tion from the ground state or else that we were exciting the protein in a heretofore un~- determined absorption band. It might be reasoned that a singlet-triplet transition which would give rise to the intense emission we observed should manifest a reasonably strong absorption and that, consequently, a spectral examination of a concentrated protein preparation might reveal this fact. Such a study did, indeed, reveal a long-wave-length maxi- mum in the absorption spectrum at 340 mz. A long-wave-length shoulder was observed in this general region for all the proteins we have examined to date, but we have not felt justified in publishing the data, for we have not assured ourselves that scattering was not considerable in our experiments. Suffice it to point out that other workers, notably Goodwin and Morton,’ have noted an “irrelevant absorp- tion” in proteins in the 340-400-my region, where the aromatic amino acids do not absorb. When, however, the lens was excited in this absorption region at 77° K., the intense blue emission had no prolonged decay time. We may conclude that, if the low-temperature, long-wave-length emission elicited in the 340-my region was origi- nating from a metastable triplet level, it was not the same level that is populated under the same condi- tions by exciting the protein in the 280-my absorption band. Homogenized lens exhibited the same spectral behavior as did the intact material. The addition of glucose to the lens homogenate did Ley ‘=. not influence the lifetime of the 77° a 400 sad K. blue emission elicited by excita- din my tion in the long-wave-length 340-my Fic. 4.—-Eemission spectrum of bovine serum al- region bumin, excited by light of different wave lengths. 10 . . . er cent aqueous solution, containing 1 per cent Albumin, Trypsin, Myosin.— plucose. T = 77° K, , Spectral data for bovine serum alb- umin, recrystallized trypsin, and myosin are represented in Figures 4-6. The emissions elicited by irradiation in the main protein absorption band around 280 3 t Emission, relative intensity 220+ 3 £ = = SiSr z : 2iOr = = 3 = eid 3 2 = 3 2 5 < : ee ¢ w s oo 2 € w o , j 800 600500 400 300 280 800 600 500 400 300 280 Ain mp din my Fic. 6.—Emission spectra of myosin Fic. 5,—-Emission spectra of recrystallized trypsin at different exciting wave lengths. | 0.24 (0.245 gm. ml,-!), 7 = 77° K. per cent aqueous solution, 7 = 77° K, 544. BIOCHEMISTRY: STEELE AND SZENT-GYORGYI Proc. N. A.S, mz were long-lived showing a considerable afterglow, while the emissions elicited by near UV around 340 my were short-lived. No afterglow could be seen visually or detected by our relatively slow instrumentation. The results are summed up in Table 1. With the exception of the long-wave-length emission of myosin, elicited by ex- citing in the 340-my region, the emission spectra for the several proteins mirror the emission properties of tryptophane more than that of phenylalanine or tyrosine. Parenthetically, the close similarity between the low-temperature emission proper- ties of the proteins and tryptophane, elicited by exciting in the absorption bands, supports the concept expressed by Teale and Weber® and ourselves,’ that energy absorbed by phenylalanine or tyrosine is transferred to tryptophane, from which molecule emission occurs. The minimum in the emission spectra around 400 my, where phenylalanine and tyrosine phosphoresce (see Fig. 2), also bears out this idea. It might be considered that the high molar absorbancy of tryptophane rela- tive to that of tyrosine or phenylalanine would result in tryptophane being the primary emitter. An examination of Table 2, where the molar ratios of the aro- matic amino acid residues in the proteins are given, shows that, when allowance is made for the ratio of molar absorbancies (approximately 1:5:35 for phenylalanine, tyrosine, and tryptophane, respectively; see Fig. 1), phenylalanine and tyrosine should be almost as efficient emitters as tryptophane. Further, when the quantum yields are considered, tyrosine has approximately the same value as tryptophane, namely, 0.21 and 0.19, respectively (Teale and Weber®). The yield for phenylala- nine is only 0.045, which may account for the nonobservance of an emission in the 300-mu region, where phenylalanine and tyrosine fluoresce. Conversely, it may be TABLE 1* Lona-Wavr- EMISSION LuenGtH ABSORPTION Lone-LivEep EvictteED BY NEAR in AQUEOUS FLUORESCENCE Puospuor- UV (& 340 mz) PROTEIN SOLUTION 298° K. 77° K. ESCENCE 298° K. 77° K Bullock lens 340 345 335 450 440 440 Bovine serum albumin 350 340 335 460 470 470 Trypsin 340 342 337 450 450 450 Myosin 340? 340 333 440 ? 420 * Numbers stand for wave lengths of maxima in mz. TABLE 2* Moves or Amino Actps 1n 105 Gm. PROTEIN PROTEIN PHENYLALANINE TYROSINE TRYPTOPHANE Bullock lens 49 35 Il Bovine serum albumin 39 29 3 Trypsin 10 23 5 Myosin 28 18 4 Insulin §2 69 0 * compiled from date taken from R. J. Block and K. W. Weiss, Amino Acid Handbook (Springfield, l.: Charles C Thomas, 1956). argued that the low quantum yield of fluorescence is evidence for an increased transi- tion probability to the triplet state, where it may be more readily degraded as heat due to the increased lifetime. We, however, are inclined to the idea that the excita- tion energy is transferred by resonance to tryptophane and then emitted.® Insulin and Tryptophane.—The close similarity in the emission properties of the Vou, 44, 1958 BIOCHEMISTRY: STEELE AND SZENT-GYORGYI 545, proteins and tryptophane led us to a comparative study of the emission behavior of tryptophane and of insulin, which contains no tryptophane. Since we have previously noted? that the blue delayed emission of tryptophane (phosphorescence) was observable only after the addition of glucose to the system (in the absence of glucose what appeared to be the same blue emission was short- lived and decayed too rapidly for us to measure the decay kinetics), we examined the emission behavior with and without glucose. Though the kinetic behavior bore out our earlier observations, the emission spectra revealed interesting new details. These studies with watery solutions of tryptophane-no-glucose and tryptophane- glucose gave the following results: 1. Tryptophane-no-glucose excited in the absorption band at 77° K. displayed no fluorescent emission band, and the blue emission band with maximum at 485 mz had no afterglow. 2. In the absence of glucose, 340-my excitation, at 77° K., elicited 435-my emis- sion, just as found for proteins; this emission also had no afterglow. 3. Tryptophane in the presence of glucose at 77° K. exhibited a fluorescent band with a maximum at 325 mp, and the blue band emitted at 440 my displayed a phos- phorescent afterglow. 4, Long-wave-length excitation of tryptophane in the presence of glucose failed to elicit the 435-my short-lived emission. In these experiments aqueous 107-* M tryptophane solutions were used. The insulin was studied at 77° K., and the exciting light was varied from 260 to 450 mz. When the protein was excited in the region of protein absorption (260-290 my), there was evidence of emission in the 290~-340-my region, which was suggestive of fluorescence from the phenylalanine and tyrosine residues, but the intensity was too low to make positive assertion. It is of interest to note, however, that we observed no 440-myu emission band, no matter what exciting wave-length region was selected. That is, phosphorescence, elicited by exciting in the region of protein absorption, was absent, as was the short-lived long-wave-length emission elicited by exciting the protein in the 340-muy region. Insulin contains no tryptophane. * This work was supported, in part, by grants from the National Institutes of Health (RG 4643 and RG 5374). + Present address: Department of Biochemistry, School of Medicine, Tulane University, New Orleans 18, Louisiana. { This work was supported, in part, by a grant from the Commonwealth Fund, the National Heart Institute (H-2042R), the Muscular Dystrophy Associations of America, Inc., the National Science Foundation, the American Heart Association, the Association for the Aid of Crippled Children, and the United Cerebral Palsy Foundation. 1 Szent-Gyoérgyi, A., Biochim. et Biophys. Acta, 16, 167, 1955. ? Steele, R. H., and Szent-Gyodrgyi, A., these Procespines, 43, 477-91, 1957. 3 Gemmill, Ch. L., Anal. Chem., 28, 1061-64, 1956. 4 Radio Corporation of America, Tube Handbook. 5 Teale, F. W. J., and Weber, G., Biochem. J., 65, 476-82, 1957. 6 For all spectra reported in this paper (with the exception of that reported in Fig, 2) quartz monochromators were used to isolate both exciting and emitted spectral regions, and nondescript transmission was no problem. 7 Goodwin, T. W., and Morton, R. A., Biochem. J., 40, 628-32, 1946. 8 Karreman, G., Steele, R. H., and Szent-Gyérgyi, A., these PROCEEDINGS (in press).