1590 PHOTOCHEMISTRY OF CHLOROPHYLL CHAP. 35 



The use of organic dyes as oxidants invites the appHcation of photom- 

 etry. French and Holt (1946) first used a visual method, determining 

 the time needed for a given suspension to "completely decolorize" a certain 

 quantity of dye. A complication arises when the absorption band of the 

 dye overlaps that of chlorophyll; it is therefore most convenient to use 

 dyes whose absorption peak is in the green. 



Photoelectric spectrophotometry permits more precise measurements 

 than visual colorimetry. In this case, a narrow band or line can be used to 

 measure the concentration of the dye in a region where the plant pigments 

 absorb only weakly or not at all. Apparatus of this type has been described 

 by Holt, Smith and French (1951) . They reported that, using the blue dye, 

 2,6-dichlorobenzenone-indophenol, the initial rate of the Hill reaction can 

 be measured within 2 min., with as little as 0.05 mg. chloroplast material — 

 a quantity which would yield only 0.1 mm.^ O2 during the same period. 

 Careful and repeatedly checked calibration (dye concentration vs. photo- 

 electric current) is required for reliable absorption measurements with 

 photoelectric cells in spectral bands isolated by filters; this calibration can 

 be avoided by the use of monochromatic light. 



Since some compounds may be reduced in light by chloroplast without 

 liberation of oxygen, photometric measurements of the Hill reaction with 

 untried oxidants must be checked to make certain that the reduction actu- 

 ally occurs at the expense of water. Washing of chloroplast material re- 

 moves water-soluble reductants (such as ascorbic acid), and thus makes 

 the photometric method less subject to errors. 



Two electrochemical methods of measuring the Hill reaction are possible. 

 One is applicable to oxidants whose reduction cause a change in acidity, 

 for example : 



(35.35) H2O + 2 Fe(CN)6-3 > 2 Fe(CN)6-' + 3^ O2 + 2 H + 



A pH meter can be used to follow the course of this reduction. This method 

 was first applied by Holt and French (1946). They checked whether any 

 pH changes occurred in light in chloroplast-free solution of Hill's reactants 

 (ferric oxalate + ferricyanide -f- ferric ammonium sulfate) and found that 

 in white light (4500 f. c.) this mixture did produce some acid — probably 

 by direct photochemical reduction of ferric oxalate; however, this reaction 

 could be prevented by the use of red light. 



A similar method was also used by Clendenning and Gorham (1950) in 

 their survey of the chloroplast preparations from different plants (sec- 

 tion B2). With some species, strong acidification occurred in the dark 

 (without liberation of oxygen). In a few cases, the pH drift in the 

 dark was in the alkaline direction (cf. section (c) below). The absence of 



