STATE OF PIGMENTS IN CHLOROPLASTS 1751 



20% soluble proteins, 10% saponifiable lipides and 20% nonsaponifiable 

 lipides (including the pigments). The pigments were: 7.5% chlorophyll, 

 0.4% carotene, 0.2% carotenoles (xanthophyll) . Electrophoretic measure- 

 ments showed that well purified chloroplastin from a gi^^en species (or two 

 closely related species) moved with a single boundary, while chloroplastins 

 from two nonrelated species (e. g., spinach and nettle) formed two sepa- 

 rate moving boundaries. The anodic velocity was relatively high: u = 

 12 X 10"^ cm.V(volt sec.) (at pH 7). The stability range was narrow, 

 pH 5.5-8.5; the velocity increased from 10.6 at pH 6 to 13.3 X 10"^ 

 cm.V(volt sec.) at pH 8.3. Sedimentation measurements showed that the 

 suspensions were not as uniform as the electrophoresis seemed to indicate. 

 Several distinct sedimentation boundaries could be observed, showing that 

 the variation in size was not continuous. Three sizes of particles appeared 

 to be present in the Aspidistra chloroplastin. Even the smallest of them 

 had molecular weights of several million. In other words, chloroplastin 

 particles were much larger and much more complex than those of hemo- 

 globin. 



Wassink (1948) suggested the term "chromophyllin" for the general type of photo- 

 synthetic protein-pigment complexes, and "chlorophyllin" for the protein-chlorophyll 

 complex in green plants. (The second term is, however, generally used for the large, 

 acid moiety of the chlorophyll molecule, cj. Vol. I, p. 439.) 



(6) Crystallized Lipoprotein-Chlorophyll 



In support of the concept of a stoichiometric protein-chlorophyll com- 

 pound, Takashima (1952) described the preparation of a crystalline chloro- 

 phyll protein from clover leaves. The procedure consisted in clarifying a 

 suspension of whole and broken chloroplasts with /S-picoline (or pyridine), 

 washing it by dialysis through cellophane into a 50-55% aqueous solution 

 of the same base, and filtering the green solution remaining in the cellophane 

 bag from the carotenoid crystals formed in it. The peak of the red absorp- 

 tion band of the filtrate was at 668 m^u, as compared to 678 mn in the orig- 

 inal suspension. Addition of dioxane until the solution contained 20% 

 dio.xane, 43% a-picoline (or pyridine), and 37% phosphate buffer (pH 7.0) 

 led — after standing for 4-7 days in the ice box — ^to the precipitation of 

 clusters of needle-like green crystals. The presence of protein in these 

 crystals was confirmed by ninhydrin, xanthoprotein and biuret reactions; 

 its molecular weight (in 55% a-pycoline) was estimated osmometrically as 

 about 19,000. It contained 0.61% P, indicating the possible presence of 

 phospholipides. Spectrophotometric estimates indicated the presence of 

 between 1.3 and 2.4 molecules of chlorophyll per protein unit of 19,000, cor- 

 responding to 7-12% by weight. It may be asked, however, whether the 

 crystal contained intact chlorophyll, or a derivative such as that known 

 to be formed by interaction of chlorophjdl with piperidine in vitro, cf. p. 

 642 and chapter 37C, section 2a. 



