45° 



H. E. DAVENPORT 



In some preliminary attempts to purify further the protein obtained 

 from pea leaves by the method of San Pietro and Lang, using salt precipi- 

 tation and electrophoresis as additional preparative steps, it emerged that 

 activity towards triphosphopyridine nucleotide (TNP) and metmyoglobin 

 were associated in the same protein fractions at all levels of purification [6]. 

 It appeared therefore that MRF is a highly purified form of PPNR. 



Methaemoglobin reducing factor as a catalyst of TPN reduction 



The activity of MRF towards TPN at two stages of purification of the 

 protein from pea leaves is shown in Fig. i. The final electrophoretic step 



200 



01 02 0-3 



mg protein added 



4 



Fig. I. Catalysis of TPN reduction by "methaemoglobin reducing factor". 

 Pea leaf protein : :• before electrophoresis ; • after electrophoresis. Reaction 

 mixtures contained (in 3 ml.) added protein as indicated, spinach chloroplasts 

 containing 0-03 mg. chlorophyll, and the following (in jumoles) : TPN, 0-4; 

 ADP, 0-5; MgCl.,, 20; Na.HPOi, 10; tris HCl buffer, pH 7-7, 150; NaCl, 40. 

 Leaf protein was omitted from the blank cell. 



gave a nine-fold increase in specific activity. With the purified protein the 

 particulate chloroplast system was here saturated by the addition of about 

 100 jug. to give a rate of reduction of TPN of 184 /nmoles/mg. chlorophyll/ 

 hr. On the basis of a molecular weight of 19 000 this would correspond to 

 the addition of 5 m/xmoles of the leaf protein. 



Comparison of metmyoglobin and TPN as hydrogen acceptors 



The relative activity of a PPNR preparation from pea leaves in cata- 

 lyzing the reduction of metmyoglobin and TPN is shown in Fig. 2. The 



