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Lab #2
Introduction to the Physical Properties of Proteins

 

 


 

Abstract

            This experiment purpose was to examine the properties of proteins. The researcher performed several experiments which included the Isoelectric Precipitation of Casein, Salting in and Salting out of Proteins, Effects of Acetone on Solutions of Albumin and Gelatin, Heat Denaturation of Albumin, and the Effect of Salt on Heavy Metal. According to research, a proteins composition and structure determines it biological properties (Kemtec Science, 2005), so the researcher was enabled to find their different properties of proteins by performing several experiments. The researcher’s goal was to find the precipitation point of the proteins by performing several experiments. The  Isoelectric Precipitation of Casein resulted in all the test tubes showing a constant pH of 5. The pH of 5 looks somewhat of a dark orange-red. The experiment involved with Salting in and Salting out of Proteins results showed that the precipitation went Gelatin first, Gelatin and Albumin second, and Albumin finally third. The third experiment required 90 drops of Gelatin and 34 drops of Albumin to reach its precipitation point. Experiment 4 results showed that Albumin coagulated at a temperature of 59 degrees Celsius. Finally, with the final experiment, the researcher found that it took 90 drops of lead Acetate in order for it to precipitate. To coagulated, “the transformation of liquid or solid into a soft semi-solid mass” (Kemtec Science, 2005).


Data/Results
Isoelectric Precipitation of Casein


Test Tube

Final pH

Max ppt

1

5

20

2

5

15

3

5

10

4

5

5

· The Max ppt stands for the precipitation point
· Each test tube was poured a certain amount of Casein
· Test tube 1: 20 drops, Test Tube 2: 15 drops, Test Tube 3: 10 drops
  Test Tube 4: 5 drops
· Acetone was added to each test tube to find out the pH
· All the test tubes resulted in a pH of 5
· Regardless of how much acetone was added to Casein Solution, it will stay at a constant pH of 5.
· pH: Dark-Orange Red.

 

 

 

Ammonium Sulphate Fractionation


Tube contains

Gelatin

Albumin

Gelatin and Albumin

1st tube to ppt

1st

3rd

2nd

Vol. of (NH4)2 SO4, which accomplishes 1st ppt

2ml

6ml

3ml

Vol. of (NH4)2 SO4, which accomplishes 2nd ppt

Already had precipitate

4ml

8ml

 

· The Gelatin solution was the first to precipitate and it didn’t need any more Ammonium sulfate.
· This chart shows how much ml of Ammonium sulfate was poured on Gelatin, Albumin, or Gelatin and Albumin for it to precipitate.
· The Gelatin didn’t need any more Ammonium sulfate because it was the first to precipitate. Yet the Gelatin and Albumin and Gelatin solution needed an additional amount of Ammonium sulfate added to it.

Acetone Precipitation


Tube contains

Gelatin

Albumin

Vol. of Acetone that causes ppt.

90 drops

34 drops

· The amount of drops may seem low because a series of things could of contribute to this experiment. The room temperature, the air, and molecules roaming around could of contributed to the outcome of the ppt.
· It required roughly 3 ml of Acetone on the Gelatin and 1ml of Acetone on the Albumin.

Heat Denaturation

Albumin coagulated at 59 degrees C.
· The researcher placed a tube filled with 3ml of Albumin and heated it in boiling water.
· The temperature of the boiling water wasn’t noted but the temperature of the test tube at which it coagulated was 59 degrees C.

Precipitation by Lead Acetate


Volume of Pb (C2H3O2)2  that causes ppt.

90 drops

· It required 90 drops of lead acetate to the albumin solution. While this experiment was being performed, the researchers slowly added drop by drop of lead acetated and mix the tube constantly.


Discussion

The researcher based his experiment on the properties of proteins. “Proteins perform a range of complex functions in nature” (Kemtec Science, 2005). That’s why this experiment revolved around the proteins and the points at which they precipitate. The researcher recorded the results of 5 experiments. The first experiment was the Isoeclectric Precipitation of Casein. The isoelectric point is when “a protein has a decreased solubility and it may precipitate” (Kemtec Science, 2005). The results showed that the pH of each test tube that contained Casein was at 5. The maximum amount of Casein that was in each tube was 20, 15, 10, 5 drops respectively. The researcher has come to a conclusion that no matter how much acetone was added to each test tube, the pH will remain acidic. Now proteins are charged molecules and the “functional groups within them are the Amino group, and carboxyl group” (Kemtec Science, 2005). Now the questions that arise are from the results is that if the acetone doesn’t affect the pH of the solution, what about the amount of casein solution added. If there is more Casein will the outcome of the pH be affected?
Now the researcher went on and performed another experiment. This experiment was the Ammonium Sulfate Fractionation. The researcher found the results to be quite interesting. The Gelatin was the first to precipitate, then the Gelatin &Albumin, and finally the Albumin. Now precipitation is the “condensation of a solid from a solution” (Kemtec Science, 2005). The second go around showed that it required 4ml of ammonium sulfate for Albumin to precipitate and 8ml of ammonium sulfate for the mixture of Gelatin and Albumin to precipitate. The researcher has found that Gelatin is much easier to precipitate than any of the other solutions used. That means Salting out was used because Ammonium sulfate has a high solubility.
The third experiment was the Acetone Precipitation. The Gelatin in this experiment required 90 drops of Acetone so that it could reach its precipitation point. The Albumin solution required 34 drops so that it could reach its precipitation point. The researcher observed the tubes carefully and watched the solutions reach the stage of precipitation. The researcher was able to interpret that the amount of drops are quite low because there could have been many factors that contributed to this. Some factors may have been the temperature of the environment around the experiment. The air itself and the particles roaming around may of lead to a mishap in the results. All of these may of lead to the problems this experiment experienced. The researcher believes that the results are accurate.
During the fourth experiment, the researcher noted the temperature at which albumin coagulated. Coagulate “is the transformation of liquid or solid into a soft semi-solid mass” (Kemtec Science, 2005). The researcher recorded the temperature at 59 degrees Celsius. The tube was placed in boiling hot water. “Heat clearly disrupts the equilibrium of these molecules” (Kemtec Science, 2005). The researcher interpreted that the temperature of the boiling water caused the solution to coagulate at the temperature that it did.
Finally, the last experiment was the Precipitation of Lead Acetate. The researcher poured 90 drops of Lead acetate and constantly mixed it. There was 3ml of the Albumin solution and it precipitated at 90 drops. There might be an error in these results because the researcher may have gone over on the amount of drops. The researcher isn’t sure why and increase in drops lead to the Albumin to precipitate when it did. Now many factors may have contributed to this. Some factors may include the room temperature or the air. The Lead Acetate is considered a heavy metal and it “precipitates proteins because the metal ions form bridges between protein molecules” (Kemtec Science, 2005). Now this happens to build up huge aggregates and the “decreases the protein-water interaction” (Kemtec Science, 2005).


References

Kemtec Science. (2005). Introduction to the Physical Properties of Proteins
In Kemtec Science. Physical Properties of Proteins
(pp. 1-5). Cincinnati, Oh: T&S Educational, Inc.