Enzyme Kinetics

Taryn Olmstead
BCHM 380 Section 7
October 16, 2017

Purpose:
The purpose of this experiment was to measure the molecular weight of unknown
proteins. This was done using a SDS-PAGE method in which the proteins are denatured and
given an evenly distributed negative charge by the use of SDS. During electrophoresis, the
proteins travel towards the positive end of the gel matrix and get sorted depending on the
fractional coefficient. The molecular weights are compared to the calibration curve created by
the molecular ladder composed of protein standards with known weights.

Methods:
Biochemistry 380 Lab Manual

Data/Calculations:

Figure 1. The results of the gel electrophoresis after staining.

Sample Well # 1 2 3 4 5
Sample Ladder Myoglobin Cytochrome Unknown A Unknown B
 Calibration Curve
y = 233711e-0.67x
- Myoglobin
100000 - Cytochrome
- Unknown A
Size (Da)

- Unknown B
10000

1000
0 1 2 3 4 5
Distance Traveled (cm)

Figure 2. Calibration curve extrapolated from the protein standard ladder results.
Distance Traveled (cm) Size (kDa)
Number of amino acid
residues*
Myoglobin 4.1 130.5 1,088
Cytochrome 4.4 207.3 1,728
Unknown A 4.0 111.9 933
Unknown B 1.8 375.6 3,130
Table 1. The distance traveled and the size of the protein samples taken from the gel analysis and
calibration curve. *The number of amino acids residues was based on the average weight of an
amino acid residue: 120 daltons.

Discussion:
Gel electrophoresis is a technique used to separate proteins based on size. The proteins
are first denatured using heat, which breaks the intermolecular bonds. The proteins must be at
their isoelectric point, ensuring that the protein has no net charge. The solution is then treated
with sodium dodecyl sulfate (SDS). SDS is an anionic detergent and surrounds the denatured
protein evenly with a negative charge. The denatured proteins are placed into the wells of a
polyacrylamide gel which has a matrix structure. A current is passed through the gel to create an
electric gradient; the samples are placed at the negative end and travel towards the positive end.
Since the proteins were treated with SDS and possess a negatvie charge, they will travel towards
the positive end while the current is present. As the proteins pass through the gel during
electrophoresis, the smaller molecules are able to navigate further through the matrix.
A protein molecular weight standard was also placed into a well to be used as a reference
for the other samples. Once the electrophoresis is complete, the bands of the samples can be
compared to the ladder that has protein standards of known molecular weights. Using the
molecular ladder, a calibration curve can be created which plots the distance migrated against the
molecular weight. The calibration curve must be graphed on a semilog graph because the data
from an SDS-PAGE does not follow a linear progression, but is rather exponential. The semilog
graph is used to create a linear trendline through the data points.
An approximation of the size of unknown proteins A and B was calculated using the
calibration curve (Fig. 2). Unknown A, which traveled a distance of 4.0 cm, had a molecular
weight of 111.9 kDa. Based on the average weight of an amino acid residue (120 Da), Unknown
A had approximately 933 amino acid residues. Unknown B on the other hand traveled 1.8 cm
giving it a calculated molecular weight of 375.6 kDa. It was also estimated to have around 3,130
amino acid residues.
There are several possible sources of error in this lab. The first error source involves the
buffer. If the buffer was not the right concentration or diluted enough, it would not function
properly. The second possible source of error was the loading of the samples into the wells. Too
much or too little of the sample would cause the band to not appear as clearly and as accurately
as it should. The final possible error source was not running the gel the proper amount of time. If
the gel was ran too long or not long enough, it is possible for the gel analysis to reveal an
inaccurate measure of the protein size.

Questions:
1. (Pg. 25) Ten ul of each sample should be placed in the well.
1. None of the sample proteins were oligomers. If they were, more than one band would
appear for that sample: one for each subunit.
2. Interferon would have migrated approximately 3.4 cm.
3. The purpose of using SDS in this experiment is to give the proteins a negative charge.
The proteins used are at their isoelectric point, meaning they have no net charge. If there
was no SDS used to provide the proteins with a negative charge, the protein samples
would not have responded to the electrophoresis and would not have moved through the
gel.