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.

Results:

After having isolated the protein and nucleic acid components from yeast cell fractionation, ascending chromatogrpahy was used to analyze the hydrolyzed and unhydrolyzed portions of each sample.

Protein Results

Results for the protein chromatograph are given below in Table 1, showing the Rf values of the hydrolyzed and unhydrolyzed samples of protein, a sample of BSA, an unknown amino acid, and five known amino acids (alanine, aspartic acid, histidine, lysine and methionine) as well.

Table 1

Solvent front(S)=10

cm

Distance Traveled (Dt)
Rf = Dt/S
1. Unhydrolyzed protein
Smear
NA
2. Hydrolyzed protein
Smear
NA
3. Methionine
7.2 cm
0.72
4 Lysine
3.6 cm
0.36
5. Unknown (A3)
2.7 cm/7.2 cm
0.27/ 0.72
6. Histidine
2.8 cm
0.28
7. Aspartic acid
4.4 cm
0.44
8. Alanine
6.2 cm
0.62
9. BSA
0.3 cm
0.03

In the above table, the Rf value of the hydrolyzed protein could not be calculated. The hydrolyzed sample developed as a large smear, indicating that the protein had been broken down successfully through hydrolysis. This can be concluded based on the fact that the BSA sample had a very low Rf value, indicative of an unhydrolyzed protein. Unfortunately, the unhydrolyzed sample of protein did not have an Rf value similar to that of BSA. An Rf value could not be calculated as the unhydrolyzed protein resulted in a smear similar to that of the hydrolyzed protein. The significance of this will be discussed later. The unknown amino acid resulted in an Rf value of 0.28/0.72. Compared with the known amino acids, unknown A3 appears to be methionine and/or histidine.

Nucleic Acid Results

Below, table 2 lists the results of the nucleic acid chromatogram along with respective Rf values. The chromatogram was run with the hydrolyzed and unhydrolyzed nucleic acid, along with unhydrolyzed calf thymus DNA, four known bases: adenine, cytosine, uracil, guanine; and an unknown base.

Table 2.

Solvent front (S) = 9.8 cm
Distance Traveled (Dt)
Rf Value = Dt/s
1. Unhydrolyzed nucleic acid
0 cm/ 1.5 cm
0.153
2. Hydrolyzed nucleic acid
0.4 cm/ 2.3 cm/ 4.3 cm
0.004/ 0.23/0.44
3.DNA
0 cm
0
4. Adenine
1.0 cm/ 4.5 cm
0.1/ 0.46
5. Uracil
4.5 cm
0.46
6. Cytosine
2.9 cm
0.30
7. Guanine
1 cm
0.1
8. Unknown (B3)
4.3 cm
0.44

In the above table, results show that the unhydrolyzed nucleic acid sample resulted in a much lower Rf value when compared to that of the hydrolyzed sample. Of the two samples, the unhydrolyzed sample appeared to have an Rf value closest to that of Calf thymus DNA. The unkown sample, B3, resulted in an Rf value most similar to that found for Uracil. Adenine produced two spots so it was not considered to be the identity of the unknown.

Glycogen Results

After fractionating out the glycogen component of the yeast cells, hydrolyzed and unhydrolyzed samples were each placed inside dialysis tubing which was then placed inside large test tubes containing distilled water. Two tests were perfromed on the fluid on the inside of each dialysis tube as well as on the fluid on the outside. For both tests, a set of controls were run which included distilled water, 5 mM glucose, 1 mM glucose, 0.1 % glycogen, and 1% glycogen.

Iodine Test: To determine the presence of the polysaccharide glycogen, iodine was added to each of the following solutions. These solutions are listed table 3 below along with the result produced upon addition of Iodine. A (+) sign indicates a positive reaction while a (-) sign indicates a negative reaction.

Table 3

Solution
Result with Iodine
1. Unhydrolyzed Glycogen (in)
Brown/red (+)
2. Unhydrolyzed Glycogen (out)
-
3. Hydrolyzed Glycogen (in)
-
4. Hydrolyzed Glycogen (out)
-
5. dH20
-
6. 5mM Glucose
-
7. 0.1mM Glucose
-
8. 0.1% Glygogen
Brown/red (+)
9. 1% Glycogen
Deep brown/red (+)

The above table shows that positive reactions resulted from the addition of Iodine in the presence of glycogen. The positive controls (8&9) reacted similarly to the result found from the solution of the unhydrolyzed glycogen lying inside the dialysis tubing.

Benedict Test: To determine the presence of hydrolyzed sugars, sugars with reducing groups such as glucose, the Benedict test was performed by adding a reagent containing a solution of sodium citrate, sodium carbonate and copper sulfate. Table 4 below summarizes the results recorded upon addition of the Benedict reagent.

Table 4

Solution
Result with Benedict Reagent
1. Unhydrolyzed Glycogen (in)
Blue
2. Unhydrolyzed Glycogen (out)
Blue
3. Hydrolyzed Glycogen (in)
Blue
4. Hydrolyzed Glycogen (out)
Blue
5. dH20
Blue
6. 5mM Glucose
Red/ Brown
7. 0.1mM Glucose
Deep Red/ Brown
8. 0.1% Glycogen
Blue
9. 1% Glycogen
Blue

The results from the Benedict test show that only the positive controls, those containing glucose, reacted with the reagent properly. A reaction was expected to occur with the hydrolyzed sample, however none occurred. The failure of the test will be discussed later.

 

Discussion

In this experiment, cell fractionation was made possible by taking advantage of specific properties of cellular components including size, charge, and differences in solubility. The first component to be obtained, glycogen, was fractionated based on its ability to dissolve in TCA solution. The glycogen was then precipitated out with cold ethyl alcohol and redissolved in HCl. Half of the sample was hydrolyzed in acid with heating. The two remaining fractions were then placed in dialysis tubing. Dialysis tubing acts as a membrane that is permeable to only small molecules such as water and glucose. The purpose of carrying out the Iodine and Benedict test was to determine if the glycogen sample had been hydrolyzed into its individual glucose sugar units. Both tests were performed on the hydrolyzed and unhydrolyzed samples. Since glycogen is a branched chain of repeating subunits of glucose its large size does not permit it to pass through the dialysis membrane. The Iodine test proved this inability of unhydrolyzed glycogen to diffuse as there was no reaction present in any of the solutions lying outside of the dialysis tubing. The reaction was only found to occur in the unhydrolyzed solution lying inside of the dialysis tubing. In addition, the Iodine test also demonstrated that the glycogen had been successfully hydrolyzed into glucose, as there was no reaction of the iodine in the hydrolyzed solution. However, the Iodine test did not give any indication of to what extent the glycogen had been hydrolyzed.

Furthermore, in the case of the Benedict test, it failed to show that the glycogen sample had been hydrolyzed fully to glucose. Since glucose is capable of diffusing through the dialysis membrane, a reaction should have occurred in the solutions of hydrolyzed glycogen, lying inside the tubing and outside. However, with the exception of the positive controls, no reaction occurred. This failure of the Benedict reagent to react with the hydrolyzed samples is most likely a result of the glycogen not being hydrolyzed fully, possibly leaving glycogen in a state of linkage involving two to three subunits of glucose as opposed to the long branching polysaccharide structure. Evidence to support this theory can also be derived from the results of the Iodine test, in which no reaction insued between the Iodine and "hydrolyzed" glycogen. Thus, the Iodine test demonstrated that the glycogen had been hydrolyzed while the failure of the Benedict test demonstrated to what extend this hydrolysis occurred. These results may also suggest that Iodine is only specific for highly branched polysaccharides, not di- or tri-saccharides.

The left over pellet from the first centrifugation of the yeast cells contained coagulated proteins and nucleic acids. The nucleic acids were separated from the proteins by adding NaCl and heating thoroughly. This caused the nucleic acids to dissociate from the protein, thus leaving the protein as a precipitate and the nucleic acids dissolved in solution. The nucleic acids were precipitated out in cold ethanol and redissolved in water. A hydrolyzed nucleic acid solution was prepared by heating in H2S04. The resulting spots on the chromatogram illustrated the effects of hydrolysis. The hydrolyzed nucleic acid solution yield three spots while the unhydrolyzed solution yielded two spots, one with an Rf value of 0 and another with an Rf value of 0.153. The three spots may be indicative of three distinct nucleotides or possibly bases. Based on the calculated Rf values of the known bases, the unknown (B3) was identified as Uracil.

The remaining fraction consisted of the protein component of the yeast cell. It was hydrolyzed using a pancreatic enzyme. The chromatograph for the hydorlyzed and unhydrolyzed did not work correctly. Both resulted in a smear after staining with ninhydrin. The results show that both samples were apparently hydrolyzed or that an error was made in applying the unhydrolyzed sample. On comparison of the unkown’s Rf value with that of the knowns, unknown A3 was identified as histidine.

 

 

References

Biology 18 B Laboratory Manual. Brandeis University. Waltham, MA. Dr. Judith Tsipis Ed, Fall 1997. Lab 2-3