FLUORESCENCE OF PIGMENTS in vivO 1881 



infiltrated) leaves immediately after a period of darkness and in the steady 

 state after the induction period is over— as if the scattering, caused by air- 

 filled spaces, increased considerably during the induction period and de- 

 creased again in darkness. In water-infiltrated leaves, or algal thalli 

 containing no air spaces, the "initial" and the "steady state" fluorescence 

 spectra show no significant differences. 



(d) Fluorescence of Protochlorophijll in vivo 



French (1954) gave spectral curves for the fluorescence of protochloro- 

 phyll in acetone solution (c/. section 4(g) above), in "chloroplastic mate- 

 rial" from etiolated leaves of barley, in inner coats of squash seeds, and in 

 the etiolated leaves after brief exposure to light. The unexpectedly com- 

 plex shapes of the curves could be interpreted in terms of two phenomena : 

 self-absorption of fluorescence, and the existence of two forms (or states) of 

 protochlorophyll — one characterized by a fluorescence peak at 635 m/x and 

 one having a peak slightly beyond 650 m/x. Self-absorption can explain 

 why, in the fluorescence spectra of tissues rich in protochlorophyll (such 

 as seed coats), both the 635 and 650 m^l bands, while present, are sub- 

 ordinated to a band at 705 m/z, which French interpreted as the sum of the 

 first vibrational fluorescence bands of both "Pchl 635" and "Pchl 650." 



The existence of two forms of protochlorophyll was first suggested, on 

 the basis of absorption spectra, by Krasnovsky and co-workers (c/. section 

 1(c) above). The locations of their long- wave absorption bands were 

 given as 635 and 645 m/i, respectively. According to Krasnovsky 's hy- 

 pothesis, "Pchl 635" is the fluorescent and photochemically active form, 

 while "Pchl 645" (or 650) is a non-fluorescent and inactive polymerized 

 form. However, the peak of the action spectrum of chlorophyll synthesis 

 in etiolated seedlings has been found at 650 and not at 635 mn (p. 1764); 

 and according to French, both "Pchl 635" and "Pchl 650" are fluorescent. 

 In this case, as in those of chlorophyll and bacteriochlorophyll, Krasnov- 

 sky's attribution of photochemical activity to the form with the higher 

 excitation level seems to be in error. The photochemically active form 

 again appears to be the one with the lower frequency absorption band- 

 perhaps merely because the electronic excitation energy is apt to be trans- 

 ferred to this form by resonance before the photochemical change has had 

 time to occur. 



The finding of two photochlorophyll modifications has solved a difficulty mentioned 

 once before— the apparent violation of Stokes' rule by this pigment whose fluorescence 

 band in vivo was first reported at 035 m/i, while its absorption band was assumed to be, 

 on the basis of action spectrum studies, at 650 m/t. 



According to French (1954), Smith has observed that "Pchl 645" is converted into 

 "Pchl 635" when freshly ground, etiolated barley leaves are heated, or allowed to stand 

 in glycerol suspension. 



