Exercise IV 



CHEMICAL COMPONENTS OF CELLS (2) 23 



protein and the protein hydrolysate. Interpret 

 your results. 



Chromatography of nucleic acid components 



Your extract of yeast nucleic acids contained 

 both RNA and DNA (see the discussion at the 

 beginning of Exercise III). The hydrolysis that 

 you have performed not only broke the nucleic 

 acid into its component nucleotides, but went 

 on to hydrolyze the nucleotides into their unit 

 components. What we are looking for now, 

 therefore, are the isolated nitrogenous bases. 

 The RNA brought in four such bases: adenine, 

 cytosine, uracil, and guanine; but since guanine 

 is relatively insoluble and difficult to detect on 

 chromatograms, we disregard it in this experi- 

 ment. Also, since DNA is present in yeast in 

 much smaller amounts than RNA, we shall dis- 

 regard its distinctive base, thymine. 



Prepare a sheet of filter paper, just as before, 

 to run on a single chromatogram your unhy- 

 drolyzed and hydrolyzed nucleic acid solutions, 

 a series of three known nitrogenous bases — 

 adenine, cytosine and uracil — and a mixture of 

 the bases. When the paper is ready, put five 

 superimposed applications of each of these solu- 

 tions at each of the labeled starting positions. 



Prepare a second quart jar just as you did the 

 other, but using as solvent acetic acid :butanol: 

 water = 15:60:25. Set this chromatogram up 

 just as you did the other, stopping it when the 

 solvent has reached 0.5 cm from the top of the 

 paper. Then remove the paper, let it dry, and 

 cut the threads. 



Instead of staining the paper this time, as we 

 did to find amino acids, we shall take advantage 

 of the fact that nucleic acids, because of the 

 nitrogenous bases which they contain, strongly 

 absorb ultraviolet light of wavelengths about 

 260 m/u. After drying your chromatogram, hold 

 it under a source of ultraviolet light. The 

 organic bases will appear as dark spots against 

 the light background. {Caution: Do not look 

 into the ultraviolet light. It is harmful to the 

 eyes. Do not expose your skin for more than 

 a few seconds.) 



Dialysis of glycogen 



Get the test tubes containing the dialyzed 

 samples of your unhydrolyzed and hydrolyzed 

 glycogen from the refrigerator. The point is 

 now to test for glycogen and its subunit, glucose, 

 both inside and outside each of the sacs. 



Pour the contents of each bag into a separate 

 clearly labeled test tube so that you now have 

 four solutions: unhydrolyzed inside, unhydro- 

 lyzed outside, hydrolyzed inside, hydrolyzed 

 outside. Pour about 1 ml of each of these solu- 

 tions into a labeled test tube. Into a fifth test 

 tube measure 1 ml of water as a blank. {Note: 

 1 ml = 20 drops.) To each tube now add 2 ml 

 of iodine reagent (iodine-potassium iodide solu- 

 tion), which stains glycogen red-brown. (Do 

 you remember the purple staining of the starch 

 grains in potato slices and Spirogyral That 

 color resulted from the fact that starch contains 

 a straight-chain blue-staining component, in 

 addition to a branched red-staining component. 

 See page 3.) 



After you have determined and recorded the 

 fractions in which glycogen is located, wash out 

 the 5 test tubes in which you did the iodine 

 test. Pour a fresh 1-ml sample of each of the 

 4 fractions and 1 ml of water into the 5 test 

 tubes. To each add 3 ml of Benedict solution, 

 swirl to mix, and place all 5 test tubes in the 

 boiling water bath for 3 min. Now compare 

 the colors. 



The Benedict test is given by all sugars that 

 contain reducing groups (aldehyde or ketone) 

 that can reduce blue cupric (Cu++) ions to red, 

 insoluble cuprous (Cu+) ions. The Benedict 

 test is negative for glycogen or starch, because 

 in them the repeating glucose subunits, each of 

 which has a potential aldehyde group (see dis- 

 cussion in III), use up these groups in the 

 glucose-glucose linkages. However, when the 

 glucose units are freed by hydrolysis, the alde- 

 hyde groups become available for reaction. 



What are your results and what do they mean ? 

 The Benedict test is not given by the sugar most 

 familiar to you, cane sugar or sucrose. Why 

 not? Make a model of sucrose. 



