16 CHEMICAL COMPONENTS OF CELLS (1) 



Exercise 



bases, and dialysis. After describing the pro- 

 cedure, we shall discuss each of these processes. 

 That discussion is an integral part of the pro- 

 cedure, so be sure to read it before you begin 

 work. We will begin with a general account of 

 the procedure, and then give explicit directions 

 in the form of a flow sheet. 



Yeast cells are enclosed in a tough, cellulose- 

 like outer wall. The wall is made of a poly- 

 saccharide called glucan, which contains only 

 glucose units, but bound to one another differ- 

 ently than in cellulose, starch, or glycogen. The 

 first operation is to break the cell walls by 

 grinding the yeast with sand in a mortar, re- 

 leasing the contents of the cells. 



Extraction and hydrolysis of glycogen 



The cell contents are stirred into trichloracetic 

 acid solution (TCA) which dissolves the glyco- 

 gen, leaving the nucleic acids and proteins as 

 solid particles in suspension. This suspension 

 is decanted from the sand, and the solid material 

 separated off by centrifuging. 



The glycogen is precipitated from the solution 

 with ethyl alcohol, and this precipitate separated 

 off by centrifuging. It is redissolved in 1 N 

 hydrochloric acid (HCl), and this solution is 

 divided in halves. One half is immediately 

 neutralized with 1 yV sodium hydroxide (NaOH), 

 to prevent hydrolysis. The other half is heated 

 for 30 to 60 minutes in a bath of boiling water 

 (i.e., at approximately 100°C), and then is neu- 

 tralized in the same way. The heating in acid 

 solution hydrolyzes the glycogen completely. 

 Each half is now placed in a dialysis sac, and 

 the sacs are suspended in test tubes containing 

 distilled water, and stored in a refrigerator until 

 next week, when we will test for the presence of 

 glycogen and glucose inside and outside the sacs. 



(NaCl), by stirring and heating in a boiling 

 water bath, leaving an insoluble residue of 

 coagulated proteins. The proteins are separated 

 off by centrifuging, and the nucleic acids in the 

 supernatant are precipitated with ethyl alcohol. 

 This precipitate is collected by centrifuging, and 

 dissolved in 1 A' sulfuric acid (H2SO4). The 

 solution is divided into halves, and one half 

 heated for 30 to 60 minutes at 100°C to hy- 

 drolyze. Then both solutions are neutralized 

 with barium hydroxide (Ba{OH)2). The salt 

 that results from the neutralization, barium 

 sulphate (BaS04), precipitates out. The reason 

 for acidifying with H2SO4 in this case, and 

 neutralizing with Ba(OH)2, is to get rid of 

 this salt on neutralization because the paper 

 chromatography of these solutions that you will 

 carry out next week goes much better in the 

 absence of salt. 



Hydrolysis of protein 



We are now ready to deal with the coagulated 

 proteins. It would take many hours of boiling 

 in strong acid or alkali to hydrolyze them. In- 

 stead, we perform this hydrolysis rapidly and 

 at room temperature by using enzymes, as do 

 living systems. We shall use a mixture of pro- 

 tein-digesting enzymes from a mammalian pan- 

 creas, which in life would have delivered this 

 mixture of enzymes to the small intestine. 



Small portions of the solid protein residue are 

 transferred into each of two test tubes. One is 

 stirred into a buffered solution of pancreatic 

 enzymes, the other into a solution containing 

 the buffer alone, to serve as control. Both will 

 be stored by your instructor until next week, 

 when their contents will be analyzed by paper 

 chromatography. 



Extraction and hydrolysis of nucleic acids 



The solid residue of the yeast cell contents 

 after the removal of glycogen contains nucleic 

 acids and proteins. The nucleic acids are ex- 

 tracted into strong sodium chloride solution 



DOING AN EXPERIMENT 



The way to go at a job such as this, whether 

 it is simple or complicated, is to read through 

 the instructions and then make a plan of attack, 

 in which you try to see yourself going through 



