62 PROCESSES OUTSIDE THE LIVING CELL CHAP. 4 



light. Similar observations were described by Macchiati (1903). However, Harroy 

 (1901), Herzog (1902), Molisch (1904) and Bernard (1904, 1905) failed to confirm them, 

 and Friedel found himself unable to reproduce his earlier positive results later in the 

 same year (190P), a failure which he attributed to the inefficiency of autumnal leaves. 



While dead or broken cells certainly are unable to carry out complete 

 photosynthesis, they may maintain a Umited capacity for evolving oxygen 

 in light (without a concurrent reduction of carbon dioxide). This was 

 first noticed in 1888 by Haberlandt and in 1896 by Ewart, who observed 

 that isolated chloroplasts (obtained by grinding leaves under water) 

 liberate a small quantity of oxygen when exposed to light. Similar 

 observations with dried leaf powders were made by Molisch in 1904. 



The matter rested there for twenty years, until Molisch came back 

 to it in 1925. He confirmed the evolution of oxygen by leaves which 

 were dried for three or four days at 30-35° C, and often kept in a desic- 

 cator for several weeks. Some leaves produced oxygen even after having 

 been heated to 84° for five hours. To obtain oxygen, dried leaves had 

 to be powdered under water and the mixture illuminated without straining. 

 Leaves killed by freezing also produced oxygen in light. Inman (1935) 

 repeated the experiments of Ewart and Molisch. Fresh leaves of 

 Trifolium repens, Zea mais and Melilotus alba were ground with sand 

 and the remaining whole cells removed by filtration. The suspension of 

 broken and unbroken chloroplasts, obtained in this way, produced 

 oxygen upon illumination. Positive results were obtained also with 

 leaves dried for several days at 30-35° before powdering. The leaves 

 lost their capacity for evolving oxygen more quickly if they were first 

 macerated and then dried, instead of drying first and powdering after- 

 wards. The alga, Nostoc, was able to evolve oxygen even after having 

 been kept in the dry state for eighteen months. Addition of protein- 

 digesting enzymes (e. g., trypsin) prevented the evolution of oxygen by 

 leaf powders. (Photosynthesis by unbroken cells was not affected by 

 trypsin.) The production of oxygen was limited to the pH range 5-7, 

 with a sharp maximum at a pB. of 5.5. Inman (1938) stressed the 

 similarity between the effects of temperature, acidity, and trypsin on 

 the oxygen evolution by leaf triturates and on the denaturation of 

 proteins, and suggested that the oxygen liberation is catalyzed by an 

 enzyme. According to Inman (1938^), the evolution of oxygen can also 

 be demonstrated with the isolated cell contents of the giant cells of 

 Nitella and Valonia macrophysa, and with press juices from clover leaves 

 and Euglena viridis. 



In all these experiments, oxygen formation could be proved only 

 by the luminescence of Beijerinck's bacteria; the rate of its evolution 

 was unknown, but certainly very small; and the whole process lasted 



