472 SMITH, KITPKE, LOEFFLER, BENITEZ, AHRNE, GIERE 



Discussion 



Witt : At what temperature were these preparations made? 



James Smith : We made them at 6°C. and of course they were done in the dark 

 in order to keep the protoohlorophyll from being transformed. 



Lxmary: As I understand it, you were al)ie to centrifuge your material to the 

 bottom of the tube in a water suspending medium. The density of the material 

 must then be greater than water and thus cannot be high in lipid-containing ma- 

 terial. 



James Smith : Yes, the density is greater than that of water. One of the proper- 

 ties we still have to determine is the density of the material so that we can get an 

 accurate determination of the molecular weight. If you assume the density to be 

 the same as that of protein, then the molecular weight comes out between 400,000 

 and 600,000. 



Kok : Have you any idea how many magnesium atoms there are in the proto- 

 chlorophyll holochrome? 



James Smith: Xo, I have no idea about the numl)er of pigment molecules nor 

 of the magnesium atoms in the holochrome. The complete experiment that I 

 have reported here was done just about 10 days before I came and we have had 

 no opportunity to analyze the .sedimented particles. We have done many partial 

 experiments and the picture is absolutely consistent in regard to the ultracentrifu- 

 gation pattern. The time of movement is very constant for this type of biological 

 material, which indicates that we are dealing with pieces of relatively constant 

 properties. 



Tolbert: After your first centrifugation at 10,000 X g, the suspension was put 

 through a French-Milner homogenizer. Why did you do that? 



James Smith : In order to break down any larger particles so as to get a yield of 

 smaller particles containing protochlorophyll holochrome. 



Tolbert: Does this imply that the holochrome protochlorophyll particle is of 

 larger size in the cell than the very small one you isolated? Could you have thrown 

 down a larger particle if .\'Ou had not put the suspension through the homogenizer? 



James Smith: It is possible that the protochlorophyll holochrome exists in 

 the leaf in organized bodies which remain intact during extraction. It was thought 

 that, perhaps by putting the extract through the French-Milner homogenizer, 

 these bodies would be disrupted and their contents dispersed in the solution. 

 Homogenization clarifies the solution to some extent, and this indicates that 

 larger particles are broken down by this treatment. Whether or not the particles 

 that are broken down are the ones that contain the protochlorophyll holochrome 

 is not known because we have never tested the solution by means of the analytical 

 ultracentrifuge before and after homogenization. 



Rosenberg: You certainly have shown very beautifully that you are justified 

 in calling this a holochrome because it shows liiological properties. Is there any 

 chance of showing some photochemical or jihysiological property that would 

 justify your calling the transformed material a "holochlorophyll"? 



James Smith: W^Il, we hope that the holochromatic chloropliyll obtained in 

 the manner described will show photochemical physiological properties. You do 

 not have to have physiological activity, however, to call it a holochrome, because 



