Xxxvi. Studies on Chloroplasts I. Separation of Chloroplasts, a Study of Factors Affecting Their Flocculation and the Calculation of the Chloroplast Content of Leaf Tissue from Chemical Analysis1

A NUMBER of references to experiments conducted with isolated chloroplasts are to be found in the literature. Englemann [1881] detected the evolution of oxygen by isolated chloroplasts and Ewart [1896] observed that they maintained their activity for some time after removal from the cell. Chloroplasts have been isolated from Stellaria media by Hill [1937] and from the leaves of the tomato plant by Granick [1937]. Noack [1927] obtained disintegrated chloroplasts by fractional centrifuging of green leaves ground with CaCO3 and Lubimenko [1929] employed autolysis to decompose cytoplasmic material. All of these workers had been concerned primarily with studies which required relatively small numbers of chloroplasts, and in most instances the details of the method of isolation were omitted. The isolation of chloroplasts in large quantities would allow the study of their chemical structure and of the distribution of metabolites in the leaf. A study of photosynthesis by isolated chloroplasts could be carried out on a large scale and the problem of the photosynthesis of sugars by the plant attacked from this angle. A procedure has been developed which gives a good yield of chloroplast substance suitable for chemical studies and also a slightly different procedure which gives morphologically intact chloroplasts. After this work was completed Menke [1938] described the separation of the leaf tissue of Spinacia oleracea into four fractions, namely, cell wall, cytoplasm, water-soluble and chloroplast substances. This method is similar in principle to the one described herein, but is less convenient to use when only the chloroplast substance is desired. He does not give evidence for the chloroplasts being morphologically intact in his preparations. More recently Granick [1938] has described a similar method which gives morphologically intact chloroplasts. Since the method of separating chloroplast granules depends on their flocculation, a study of the factors influencing the rate and degree of flocculation has been made. A know,ledge of the percentage of the dry weight of leaf tissue which is contributed by chloroplasts is desirable in any study of the distribution of metabolites in leaf tissue. The chloroplast content of various leaves has been determined directly by weighing the chloroplast substance separated from a known amount of leaf tissue and indirectly by calculation from the analyses of