BIOSYNTHESIS OF CHLOROPHYLL; THE PROTOCHLOROPHYLL 1765 



observed oxygen liberation was <2.5% of that calculated on the assump- 

 tion that the reduction occurs at the expense of Avater (protochlorophyll + 

 H2O ->- Chi + O2). Under similar conditions, in leaves which contained 

 some chlorophyll, oxygen evolution from photosynthesis could be easily 

 observed; this shows that the lack of oxygen production in etiolated leaves 

 was not due to immediate re-utilization of this gas by the cells. The con- 

 clusion is thus justified that the photochemical conversion of protochloro- 

 phyll into chlorophyll is not coupled with the oxidation of water. (It 

 therefore cannot represent the oxygen-liberating step in photosynthesis, 

 as has been occasional^ suggested.) 



Smith and Benitez (1954) inquired into the temperature dependence of 

 the initial protochlorophyll -»► chlorophyll conversion in light, over a wide 

 range, from -175° to -^55° C. They noted that heating above 40° C. 

 progressively destroyed the capacity for conversion and suggested that 

 this indicates the denaturation of a protein (to which protochlorophyll can 

 be assumed to be attached in vivo, and which may be essential for the 

 transformation; dissolved protochlorophyll is not converted to chlorophyll 

 by illumination). 



Cooling below 40° C. slowed down the photochemical conversion. 

 This, and the fact that the reaction followed a bimolecular law (strictly at 

 589, 579 and 546 m/i ; less strictly at 436 m^u) indicated interaction between 

 an excited and a normal protochlorophyll molecule. (Proportionality of 

 the rate Avith the first power of light intensity excludes a reaction between 

 two excited molecules.) Perhaps the reaction is a dismutation with one 

 protochlorophyll molecule being reduced and one oxidized, but no oxidation 

 product has yet been observed. 



The temperature curve can be followed down to —80° C, at which 

 temperature the conversion is still appreciable in both rate and total 

 amount. It becomes unobservable at —195° C. Tha\ving after freezing 

 destroys the capacity for conversion; slow passage through the freezing 

 zone damages it. In order to measure the rate at —10° or —20° C, the 

 leaves must be snap-freezed at —80° C. and then warmed up. 



Some experiments were made by Smith and co-workers on the rate 

 of protochlorophyll formation by barley seedlings in the dark. A sigmoid 

 curve was obtained, almost no protochlorophyll being formed in the first 

 three days of germination, an accelerated formation occurring between 

 the third and the sixth day, and the synthesis slowing down after the sixth 

 day. The development of carotenoids followed a similar course. 



Godnev and Terentjeva (1953) found that the conversion of protochloro- 

 phyll to chlorophyll can be achieved, in etiolated corn seedhngs, instead of 

 by exposure to light, also by infiltration with an extract from pine seed- 

 lings (conifers, like algae, can form chlorophyll in darkness). 



