PIGMENT-PROTEIN SUSPENSIONS AND SOLUTIONS 387 



Aspidistra leaves, recommended by Lubimenko.) After separation from 

 the dissolved cytoplasmic proteins (by centrifugation or filtration through 

 paper pulp), the green chloroplast matter was resuspended by Smith in 

 pure water. It was precipitated by heating above 60° C. or by add- 

 ing half-saturated ammonium sulfate, and coagulated by dilute acids 

 (pH 4.5). The latter do not convert the chlorophyll in these suspensions 

 into pheophytin (as they would in absence of the protein), so that the 

 acid precipitate gives a pure green solution upon resuspension. However, 

 it cannot be resuspended in pure water, but only in dilute alkali (pH 9) . 

 The absorption spectrum of the suspension (reproduced in Vol. II, Chap- 

 ter 21) was similar to that of the living leaf. Smith called his prepara- 

 tions "nonfluorescent," while Noack (1927), Stoll and Wiedemann (1938), 

 and Fishman and Moyer (1942) have described similar suspensions as 

 "weakly fluorescent." 



Smith's turbid chlorophyll-protein suspensions were clarified instan- 

 taneously by various detergents (digitonin, sodium dodecyl sulfate, so- 

 dium desoxycholate or bile salts) and thus probably converted into true 

 macromolecular solutions. (The detergents have the same effect on 

 colloidal solutions of pure chloroph3dl.) The clarified solutions have 

 been studied by Smith and Pickels (1940, 1941) by means of the ultra- 

 centrifuge. Their properties depend on the nature of the detergent. 

 Digitonin (as well as sodium desoxycholate and bile salts) splits the 

 pigment from the protein; the pigments sediment together with the 

 digitonin micelles; while the pigment-free protein forms another bound- 

 ary, corresponding to a sedimentation constant of 13.5 X 10~^^ and a 

 molecular weight (calculated from Stokes' law) of approximately 265,000 

 (that is, about one-half of Wyckoff's value). Sodium dodecyl sulfate, 

 on the other hand, leaves the pigment attached to the protein, but 

 splits the latter into smaller units. Furthermore, in acid solution, 

 chlorophyll becomes converted into pheophytin, showing that dodecyl 

 sulfate destroys the protection which magnesium enjoys in the natural 

 chloroplastin complex, even though large fractions of the original protein 

 molecule remain attached to the pigment. 



The absorption spectrum of the colloidal chlorophyll solutions clarified 

 by detergents remains similar to that of the living cell (except in the far 

 red; cf. Chapter 21, Vol. II). They are nonfluorescent. 



All these experiments, while confirming the association of chlorophyll 

 with the chloroplast proteins, do not prove the existence of a chlorophjdl- 

 protein compound of a constant stoichiometric composition, comparable 

 to hemoglobin. Determinations of the average chlorophyll-protein mass 

 ratio in chloroplasts {cf. page 389 et seq.) prove that there is not enough 

 protein available to provide each chlorophyll molecule with a "protein 

 unit" of the same size as in hemoglobin. 



