The Proteins of Green Leaves. VI. Centrifugal Fractionation of Tobacco Leaf Homogenates and Some Properties of Isolated Chloroplasts.

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Abstract
Tobacco leaf homogenates were separated into fractions by differential centrifugation, and the fractions analyzed for protein nitrogen, chlorophyll, nucleic acid, catalase and cytochrome oxidase. From the data obtained, the following classes of particles may be inferred to be present: nuclei; chloroplasts; small particles (presumably mitochondria) rich in catalase, cytochrome oxidase and nucleic acid; and very small particles poor in catalase but otherwise similar to the presumptive mitochondria. Of these particles, only the chloroplasts have been purified thoroughly. Purified chloroplasts have only extremely small amounts of the two enzymatic activities looked for and of nucleic acid; and the presence of these may therefore be taken as evidence for impurities in a chloroplast preparation. Supporting evidence is seen from the impure centrifugal fractions: in these, the enzymes and nucleic acid are diluted on a mg protein nitrogen basis in the fractions where chloroplasts are the most concentrated. Chloroplasts are found to be disrupted by grinding the leaves with a mortar and pestle, whereas grinding with a blendor leaves most of them intact. The reverse situation obtains for nuclei. Having obtained pure chloroplasts, it is possible to measure the amount of chloroplast fragments in the smaller fractions, and by difference determine the amount and specific activities of non-chloroplast particles in each fraction.