1554 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



periments did not confirm Arnon's hypothesis that chloride protects chloro- 

 plasts from light injury, since to restore the photochemical activity of 

 chloride-free chloroplasts it proved enough to add chloride after the pre- 

 illumination. The chloride thus acts directly on the photochemical reac- 

 tion. Its effect was found to be proportionately the same at all light in- 

 tensities. The pH optimum of the Hill reaction was found to shift in the 

 presence of chloride to higher values (from pYl 6.5 to 7.1 with Hill's mix- 

 ture and 7.5 with quinone) (fig. 35.22E). Gorham and Clendenning em- 

 phasized the similarity of the anion effect on the Hill reaction with the ef- 

 fects observed in other enzymatic reactions, such as starch hydrolysis by 

 dialyzed amylase or respiration of washed root discs, and suggested that 

 the same explanation should apply to all of them. 



The fact that chloride stimulation of chloroplast activity occurs only at 

 pR > 6 (fig. 35.22E) may explain why no chloride influence could be found 

 in the Hill reaction (as well as in photosynthesis) of whole Chlorella cells 

 (c/. part C below). 



To sum up, the nature of the salt effect on the Hill reaction is not yet 

 clear; perhaps, at least in part, this effect is due to coagulation of smaller 

 colloidal particles into larger ones which are more active photochemically 

 {cf. next section) and less subject to deactivation {cf. section (b)). 



(d) Fractionation of Chloroplast Material 



Since the Hill reaction permits an important part of the photosynthetic 

 apparatus to be separated from the living cell, biochemists would like to do 

 with this part what they did so successfully with many other enzymatic 

 systems— fractionate it by methods of protein chemistry and concentrate 

 the photocatalytic principle in a small fraction ; or, if the catalytic system 

 is complex, separate it into its components. So far, this undertaking has 

 not been successful. Several difficulties are responsible for the lack of 

 progress. 



In the first place, the chloroplast-protein material is not water-soluble. 

 In other words, it does not fall apart, in contact with water, into more or 

 less uniform building stones of macromolecular size. By mechanical frag- 

 mentation, chloroplastic matter can be converted into a water-born sus- 

 pension; and the fragm^ents can be disintegrated still further by one of 

 the methods mentioned in section 3(a). Sols with colloidal particles of 

 more or less uniform size can then be obtained by fractional centrifugation. 

 However, there is no reason why such particles should consist of chemical or 

 functional units; it is not even certain that they are identical in composi- 

 tion and structure (and not merely in size) . 



This difficulty is common to all work with water-insoluble proteins. 

 The chloroplastic matter, in addition to containing insoluble proteins 



