Effect Of Ph On Fluorescence

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Vol. 7I


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Effect of pH on Fluorescence ofTyrosine,
Tryptophan and Related Compounds
Department of Biochemi8try, Univer8ity of Sheffield
(Received 28 July 1958)

Unlike the absorption spectrum, the fluorescence
spectrum of proteins is not the simple sum of the
contributions of the aromatic amino acids in
neutral water solutions (Teale & Weber, 1956).
A study of the behaviour of substituted aromatic
amino acids andsimple peptides was undertaken in
an attempt to elucidate phenomena contributing to
the fluorescence of proteins. Ultraviolet excitation
was used to reach the lowest excited singlet state of
the compounds and interest was concentrated in
energy loss by processes competing with fluorescence, i.e. a reduction in the quantum yield of
fluorescence. The changes in fluorescence yield
withhydrogen-ion concentration were studied.

The fluorescence-excitation spectra were determined by
means of the apparatus described by Teale & Weber (1956).
Briefly, the principle was to irradiate a cell containing the
test solution with monochromatic light and to view the
light emitted at right-angles to the incident light by means
of a photomultiplier. A filter to block anyscattered
exciting light was interposed between the cell and the
photomultiplier. The photomultiplier was an EMI 6255 or
27M3 Mazda. A Perspex filter absorbing light of wavelengths less than 300 m,u was used to separate exciting light
and fluorescence.
Cells of black mat glass with crystalline-quartz windows
attached by a non-fluorescent cement were used to reduce
to a negligible value cellfluorescence with irradiating light
of wavelengths 230-260 m,u (Weber & Teale, 1958).
To eliminate the effect of pH on absorption of the tyrosine derivatives the illuminating wavelength was chosen at
the isosbestic point (Fig. 1).
Hydrochloric acid was used to adjust the pH of the
aqueous solutions on the acid side of neutrality and sodium
hydroxide solution for the alkaline pH values. Nobuffer
solutions were used. The instrument used to measure the

pH values was a Cambridge Pye pH meter with ? glass and
a standard calomel electrode. The pH scale was standardized according to the B.S. specification.

The methyl esters of tyrosine and tryptophan were prepared by the following modification of the method of
Brenner & Huber (1953). A mixture of thionyl chloride,methanol and tyrosine (in the molecular proportions
1:8:1) was allowed to stand for 12 days at room temperature. The methanol was removed under reduced pressure,
the residue dissolved in water, and adjusted to pH 6 and
extracted with ether. A white crystalline material was
obtained (m.p. 134-1380 uncorr.), which on a paper chromatogram with butanol-acetic acid-water solvent mixture
(4:1:5) had Rp0-585 compared with 0-3 for tyrosine. In the
preparation of the tryptophan methyl ester the thionyl
chloride, methanol, tryptophan mixture (in the molecular
proportions 1:8:1 as described above) was maintained at
00 for some hours until a heavy salmon-pink precipitate
was formed from the dark-red solution. The methanol was
removed as described above and an ether extraction performed. Upon...
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