Int. J. PeptideProtein Res. 11,1978,91-92 Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

T H E “ANOMALOUS” 304nm EXTREMUM IN T H E T H E R M A L P E R T U R B A T I O N OF T R Y P T O P H A N

Absence of a Fluorescence Artifact

JAKE BELLO and HELENE R. BELLO

Dept. o f Biophysics, Roswell Park Memorial Institute Buffalo, New York, U.S.A.

Received 26 July, accepted for publication 12 September 1977

Thermal perturbation spectra of a-chymotrypsinogen with and without the fluorescence quencher Eu(N03)3 show that the “anomalous” 305 nm extremum (presumed to arise from tryptophan residues) is not a fluorescence artifact. A similar result was obtained for p-acetyltryptophan amide in 1 3 . 6 M potassium formate.

Key words: fluorescence; thermal perturbation.

Thermal perturbation (TP) spectra of tryptophancontaining proteins sometimes show extrema at about 300-305nm in addition to the extrema at about 292-295nm shown by model compounds (Bello, 1970; Nicola & Leach, 1976). In solvent perturbation spectra similar “anomalous” extrema are sometimes seen at about 305nm. Nagy (1977) has suggested that the 305 nm extremum in solvent perturbation is an artifact arising from differen- tial fluorescence between the aqueous and the mixed solvent.

Nagy’s report directed our attention to the possibility of a fluorescence artifact in TP spectra. For crchymotrypsinogen (Fig. 1) AA3w is -0.007 for a solution for which A3* is 0.1. An Am of 0.1 corresponds to 20% absorbance (80% transmission). The quantum yield of crchymotrypsinogen is 0.075 (Long- worth, 1971), and the fluorescence is 25% greater at 4” than at 26”. Therefore, the differ- ence in fluorescence between 4” and 26” equals about 0.4% of the incident radiation. Taking I. in the equation AA = log Io/I as the radiation transmitted by the 26” sample and I as that transmitted by the 4” sample, AA is about

-0.004, or one-half of the observed AA3w. The value of 0.004 is an upper limit which must be drastically reduced when considering that only a small fraction of the emitted radiation

125 1

-750 1 I

290 310 330 350

Wavelength, nm FIGURE 1 Thermal perturbation spectra of Eu(NO,), and a- chymotrypsinogen. A: Eu(NO,),, 0.025 M; B: IY- chymotrypsinogen, 1 mg/ml; C: Eu(NO,), and IY- chymotrypsinogen. All solutions are in 0.05 M ammonium acetate, pH 6.4.

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J . BELLO and H.R. BELLO

can fall onto the photomultiplier cathode because of the geometry of the optical path. Thus, it appeared unlikely that the 304nm extremum is a fluorescence artifact.

Nevertheless, we did some experiments to test this further. We examined cuchymotryp- sinogen, which has a 304nm extremum in its TP spectrum. We measured the TP spectrum of cuchymotrypsinogen in the presence and ab- sence of the fluorescence quencher E u ( N O ~ ) ~ . This quencher has a TP spectrum with a “Window” in the required wave length range, 300-310nm (Fig. 1). With 0.025 M E u ( N O ~ ) ~ the fluorescence of cuchymotrypsinogen at 304nm was reduced by 74%, but the TP spec- trum at 304nm was not affected significantly. The magnitude of AA of spectrum C at 304 nm, is close to that of spectrum B (taking into account the small positive AA of E u ( N O ~ ) ~ at this wave length). The residual difference (0.0007) is within the limits of error of the method. With half the concentration of E u ( N O ~ ) ~ , 0.0125 M, fluorescence was reduced by 59%, with no effect on the 304nm extremum of the TP spectrum.

E u ( N O ~ ) ~ , 0.025 M, also decreased the fluor- escence of P-Ac-Trp-NHz by 75%. Although P-Ac-Trp-NHz in water does not have a 305 nm TP extremum, it does have one when dis- solved in very concentrated solutions of potas- sium formate (Bello, 1970). E u ( N O ~ ) ~ , 0.025

M, reduced the fluorescence of P-Ac-Trp-NHz in 13.6M potassium formate by 60%, with no effect on the TP extremum at 305 nm.

Thus, experiment shows that the 305 nm extrema in these TP spectra are not fluor- escence artifacts.

It is of interest that E u ( N O ~ ) ~ quenches the fluorescence of p-Ac-Trp-NHz and cuchymo- trypsinogen to the same extent (74-75%) suggesting that nearly all of the fluorescence of the protein arises from exposed tryptophan residues.

REFERENCES

Bello, J . (1970) Biochemistry 9, 3563-3568 Nicola, N.A. & Leach, S.J. (1976) Int. J. P e p . Prof.

Nagy, B. (1977) Fed. Proc. 36,813 Longworth, J.W. (1971) in Excited States of Proteins

and Nucleic Acids (Steiner, R.F. & Weinryb. I., eds.), p. 434 , Plenum Press, New York.

Res. 8,393-415

Address: Jake Bello, Ph.D. Roswell Park Memorial Institute 666 Elm Street Buffalo, New York 14263 U.S.A.

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