Fluorescence-Spectrum Curves of Chlorophylls, Pheophytins, Phycoerythrins, Phycocyanins and Hypericin.

Abstract

Any quantitative investigations of fluorescence phenomena more or less presuppose some information as to the spectral emission curve of the substances under investigation. Knowledge of the shape of the slpectral emission curves of pure substances is necessary to various degrees of accuracy for the identification of pigments by fluorescence, for the detection of fluorescent impurities in mixtures, for the appropriate selection of filters or photocells to measuire the fluorescence intensity, and for planning experiiments so as to avoid reabsorption of the fluorescence within the sample. Reliable curves for the pure substances are particularly necessary for the analysis of the fluorescence spectra of complex fluorescent mixtures or of living cells. The fluorescent properties of chlorophyll have been investigated from many points of view since the fluorescence of chlorophyll was discovered by Brewster in 1834. Stokes, who discovered the basic principles of fluorescence, also found that chlorophyll fluoresces in leaves. In spite of the widespread occurrence in nature of chlorophyll and its scientific importance we know of only one investigation, Zscheile and Harris (32), reporting the precisely meastured fluorescence spectra of pure chlorophylls a and b. The fluorescence spectrum of bacteriochlorophyll in solution has been measured by Vermeulen, Wassink, and Reman (27) and in the intact bacteria by Duysens (9). The present paper revises slightly the calculations upon which Zscheile and Harris based their curves and presents precise curves for other plant pigrments, particularly those concerned in the process of photosynthesis. The yield of chlorophyll fluorescence in polar solvents has recently been found by Livingston and Forster (18) to be 25 in contrast to the older accepted value of 10 %. The low fluorescence in pure non-polar solvents and the enhancement of its intensity by traces of polar solvents has been studied by Li-ingston, Watson, and McArdle (19). Various aspects of chlorophyll fluorescence have been reviewed by Rabinowitch (22), De Ment (5), Pringsheim (21), and F6rster (10). Fluorescence spectroscopy provides a very sensitive method for the identification and quantitative determination of the various chlorophylls and other fluorescent substances in plant extracts (13, 14, 15, 23, 29, 30). Fluorescence spectroscopy of photosynthetic pigments in live plants has been used in studying energy transfer from one pigment to another by Dutton, Manning and Duggar (7), Wassink and Kersten (31), van Norman, French and Macdowall (26), French and Young (12), and Duysens (8, 9). The interpretation of the fluorescence spectra of li-ve plants containingi, several fluorescent pigments an(d the use of these spectra in the study of energy transfer depend upon a knowledge of the fluorescence spectra of the individual pigments. Most of the previous investigators of the spectral characteristics of the p)hotosynthetic pigments used visual or photographic observation of the wavelength position of the peaks. Much valuable exploratory work of this type was done by Dh6re and his collaborators (6). Visual anid photographic methods are reasonably adequate for measuring the wavelength of major emission bands but are poor for the lesser maxima and often entirely inadequate for those bands that show only as a shoulder on a more intense band. Careful photometry of photographic fluorescence spectra, even when combined with the best calibration procedures, is less precise than the direct photoelectric spectrophotometric measurement of fluorescence curves except when the fluorescence is so weak that only prolongedl photographic exposures can detect it. Furthermore, photographic and visual procedures may show distorted positions of the wavelength of the peaks duie to the characteristics of the photographic emulsion or of the human eye. Considering the uitility and significance of precise fluorescence curves of biologically important pigments, it is surprising that so few of them have been measured. The chance to make such measurements arose through fortunate coincidences: the pure chlorophylls were on hand through the work of Smith and Benitez (23), the pure phycobilins were prepared for another investigation by Blinks and Airth at the Hopkins Marine Station, and the equiipment wstl on lhand from previous work (11, 12, 15, 28, 29, 30).