1. Aqueous extracts of spinach and Aspidistra leaves yield highly opalescent preparations which are not in true solution. Such extracts differ markedly from colloidal chlorophyll in their spectrum and fluorescence. The differences between the green leaf pigment and chlorophyll in organic solvents are shown to be due to combination of chlorophyll with protein in the leaf.
2. The effect of some agents on extracts of the chlorophyll-protein compound has been investigated. Both strong acid and alkali modify the absorption spectrum, acid converting the compound to the phaeophytin derivative and alkali saponifying the esterified groups of chlorophyll. Even weakly acid solutions (pH 4.5) denature the protein. Heating denatures the protein and modifies the absorption spectrum and fluorescence as earlier described for the intact leaf. The protein is denatured by drying. Low concentrations of alcohol or acetone precipitate and denature the protein; higher concentrations cause dissociation liberating the pigments.
3. Detergents such as digitonin, bile salts, and sodium desoxycholate clarify the leaf extracts but denature the protein changing the spectrum and other properties.
4. Inhibiting agents of photosynthesis are without effect on the absorption spectrum of the chlorophyll-protein compound.
5. The red absorption band of chlorophyll possesses the same extinction value in organic solvents such as ether or petroleum ether, and in aqueous leaf extracts clarified by digitonin although the band positions are different. Using previously determined values of the extinction coefficients of purified chlorophylls a and b, the chlorophyll content of the leaf extracts may be estimated spectrophotometrically.
6. It was found that the average chlorophyll content of the purified chloroplasts was 7.86 per cent. The protein content was 46.5 per cent yielding an average value of 16.1 parts per 100 parts of protein. This corresponds to a chlorophyll content of three molecules of chlorophyll a and one of chlorophyll bfor the Svedberg unit of 17,500. It is suggested that this may represent a definite combining ratio of a and b in the protein molecule.
