CHEMICAL COMPONENTS OF CELLS: 

 MACROMOLECULES OF YEAST AND 

 THEIR SUBUNITS (2) 



^4S'* 



(Reading: W. H. Stein and S. Moore, "The Structure of Proteins," Sci. Am. 

 192, No. 5, 36-^1, May 1955.) 



Paper chromatography separates compounds 

 on the basis of their different rates of migration 

 on filter paper (cellulose). The rates of migra- 

 tion depend upon the solvent which is flowing 

 up or down the paper, and on the relative 

 strengths of adsorption which hold the mole- 

 cules more or less tightly to the paper. Some 

 substances can be separated fairly well in dis- 

 tilled water, but mixtures of water with various 

 organic solvents are usually more effective. 

 Depending upon whether the solvent flows down 

 or up the paper, one speaks of descending and 

 ascending chromatograms. We will use ascend- 

 ing chromatograms. 



Chromatography of amino acids and protein 

 hydrolysates 



In this experiment you will chromatograph on 

 a single sheet of filter paper your unhydrolyzed 

 protein, the protein hydrolysate you have pre- 

 pared, an unknown amino acid, and five known 

 amino acids (alanine, aspartic acid, histidine. 



lysine, and methionine). All these things will be 

 lined up as though getting ready for a race. 

 The measurement will consist in permitting 

 them to run for a time, and then finding how 

 far each has gone. The ratio of the distance a 

 given substance has moved from the starting 

 point to the distance traveled by the solvent 

 front is called the Rp. Two substances having 

 the same Rp, particularly when this has been 

 measured in a variety of solvents, are probably 

 identical; and the Rp of any known substance 

 under particular conditions is an important 

 identifying property. 



Lay a square piece of filter paper, 12-cm 

 across, on wax paper, and draw a fine line, with 

 a lead pencil, parallel to and 1.5 cm from one 

 edge. This will be the bottom of your chromato- 

 gram. (Note: Avoid excess handling of the 

 filter paper, since your hands may contaminate 

 it with amino acids. Touch it only at the edges.) 

 On this line mark pencil dots, 13 mm apart, 

 starting 20 mm from one edge. These are to 

 indicate the positions for placing your samples. 



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