634 



LIGHT AND LIFE 



and mixed. The optical density of the mixture is measured against a 

 bhuik which contains buffer, nucleotide, and chloroplasts before and 

 after illumination for a certain period of time. The increase in optical 

 density, calculated from the two measurements, serves as a measure 

 of tW. enzymatic activity. 



It should be noted that both the blank and the reaction cuvette 

 are illuminated. Since the blank contains everything but enzyme, the 

 data obtained in this manner are corrected for the slow endogenous 

 rate of TPN reduction by chloroplasts alone in the absence of enzyme. 



One unit of enzyme is defined as the amount that produces a 

 change in optical density of 1.0 in 10 min at 340 m^ when the reaction 

 mixture contains 100 ^g chlorophyll per 3 ml. This unit of enzyme 

 corresponds to the reduction of 4.8 /i,moles pyridine nucleotide per 

 mg chlorophyll in 10 min. 



Purification of Enzyme 



The purification of the enzyme has been described in part previ- 

 ously (19, 20, 22) and involves the following steps: (1) preparation 

 of a crude homogenate from spinach leaves; (2) fractionation with 

 acetone; (3) precipitation with protamine sulfate and extraction of 

 the residue; and (4) treatment with a mixture of bentonite and 

 Dowex-1 formate followed by concentration of the enzyme with 

 acetone. 



A summary of the average yields and purifications obtained at each 

 step for a number of different preparations of the enzyme is presented 

 in Table 1. It is seen that the over-all recovery is 32 per cent and 

 the increase in specific activity is about 200-fold. 



Properties of Enzyme 

 Effect of Enzyme Concentration 



Fig. 1 illustrates the relationship of the concentration of enzyme to 

 the amount of TPNH formed. It can be seen that the test is linear 



TABLE 1 

 Summary of Enzyme Purification 



Fraction 



Total units 



Specific Activity 

 units/mg protein 



Crude Homogenate* 

 Extract of acetone precipitate 

 Protamine sulfate 

 Dowex-bentonite concentrate 



r)000 



5150 

 3500 

 1600 



0.2 



1 . 5-2 . 5 



8-12 



35-50 



Per cent 

 Recovery 



103 

 70 

 32 



Spinach leaves, 1000 g, served as the starting material. 



