Introduction:
Yeast cells are simple, unicellular, eucaryotic organisms belonging to the Fungi kingdom. These cells are particularly important as tools for research because they share structural and compositional similarity with cells of higher organisms. This experiment makes use of these similarities to study individual macromolecular components found within all living cells. Through this experiment we will learn the basic sub-units that make up each of these macromolecules while also learning some of their important structural characteristics.

This experiment will consist of two parts. The first of which will divide the yeast cells into three of its major macromolecular components: nucleic acids, proteins and polysaccharides. These components are large macromolecules that are quite unique in their composition, structure and function. However, they share a common feature as each macromolecule is composed of repeating subunits, characteristic of the macromolecule. The subunits are linked together by a bond between two adjacent subunits, formed by the loss of water (condensation). Thus, macromolecules can be broken down by the addition of water across the bond, in a process known as hydrolysis. This process was used in the experimental procedure to allow analysis of each individual macromolecule in its subunit form. Proteins are hydrolyzed into amino acids, nucleic acids are hydrolyzed into sugar, base and phosphate, and polysaccharides are broken down into simple sugars.

In the second part of the experiment, the principle method of chromatography was used to analyze the macromolecules isolated in part one of the experiment. With this technique, individual molecular species were separated from one another. This separation technique was performed for the protein and nucleic acid components. In addition, for each case, a series of knowns including one unknown sample was run. Thus, in the case of the nucleic acid, four nitrogenous bases and one unknown base was run, and in the case of the protein, five known amino acids and one unknown amino acid was run as well. In analysis of the polysaccharide component, two methods were utilized in the detection of simple sugars (glucose) and branched sugars (glycogen). The polysaccharide component was first subjected to dialysis, a method of further fractionating the simple sugars from the branched sugars. An Iodine test was used to detect the presence of unhydrolyzed glycogen and the Benedict test was used to detect the presence of hydrolyzed glycogen (glucose).

The results of the experiment show clearly the chemical bond similarities that exist between proteins, nucleic acids and polysaccharides. Each was hydrolyzed successfully into its individual sub-units. The nucleic acid chromatogram resulted in two distinct patterns for the hydrolyzed and unhydrolyzed nucleic acid. A separation of three spots occurred for the hydrolyzed sample. The protein chromatogram was also successful in demonstrating a separation of the individual subunits of DNA or RNA. Unknown samples for each were identified (A3 = methionine and histidine, B3= Uracil). The Iodine test indicated the presence of glycogen in only the unydrolyzed dialyzed sample inside the tubing while the Benedict test failed to indicate the presence of fully hydrolyzed glycogen.

Methods:

In the first part of the experiment, yeast cells were collected and carried through a process of cellular fractionation to yield the polysaccharide glycogen, nucleic acids, and proteins. This fractionation process is outlined on pages 52-58 of the lab manual. No changes were made to the outlined procedure. In each case, a portion of each cellular component was put through an additional hydrolysis step thus breaking down the specific component into its basic subunits. In addition, for preparation of the glycogen samples, both hydrolyzed and unhydrolyzed samples were placed in dialysis tubing for further fractionation.

In the second part of the experiment, the protein and nucleic acid samples were analyzed using ascending chromatography. Along with the hydrolyzed and unhydrolyzed samples, a series of knowns including one unknown were run as well. The nucleic acid chromatorgram was visualized under UV light while the protein chromatogram was visualized using ninhydrin-acetone, a powerful oxidizing agent that causes a color reaction. The procedure can be found on pages 60-65 of the lab manual. Both the inside and outside portion of the dialyzed samples of glycogen were analyzed using the Iodine test and the Benedict test. In the presence of glycogen, iodine produces a deep reddish-brown color. The Benedict test on the other hand turns reddish brown only in the presence of reducing sugars, such as glucose.