Samuel Roberts

MOL 214 Section B09

TA: Ben Lovett

21 February 2018

Lab 2: Myoglobin

1. Purpose:

The purpose of this experiment is to investigate the three-dimensional structure,

specifically the tertiary and secondary structures, of holomyoglobin and apomyoglobin and test

the difference in the susceptibility of these two forms to digestion with trypsin, a serine protease

that cuts proteins on the right side of lysine and arginine (unless it is followed by a proline).

Holomyoglobin and Apomyoglobin are variations of myoglobin, a small protein found in muscle

cells. While holomyoglobin contains a heme group, which is a functional group that is added to

the protein after it has been synthesized (also called prosthetic group), apomyoglobin does not.

According to structural studies of holomyoglobin and apomyoglobin, we know that a specific

alpha helix of apomyoglobin is affected by the absence of a heme group, which causes the

protein to be relatively unstable and more susceptible to digestion with protein. In this

experiment, we use both apomyoglobin and holomyoglobin to compare their secondary and

tertiary structures and understand how the differences in their structures affect their likelihood of

being digested by a protease (which is an enzyme that breaks proteins) like trypsin. We will also

be able to identify the helix that becomes unfolded after the heme group is removed by analyzing

the “cut” sites of the myoglobin’s primary structure. A process called SDS-page is used to

separate a mixture of proteins and identify a specific protein by finding its molecular-weight,

based on how much distance it migrates by comparing it to a molecular weight “ladder” made up
of known standards. Through this process, it displayed clearly which structure was digested by

trypsin because two bands were clearly seen, showing the cut sites that were exposed to the

protease.

2. Data:

Figure 1— SDS-PAGE of Protein Standard, Holomyoglobin and Apomyoglobin with and

without protease.

Lanes: 1. 2. 3. 4 5. 6. 7. 8. 9. 10.

Lane 1 Lane 2 Lane 3 Lane 4 Lane 5

Protein
Sample Standard Holo Holo and Protease Apo Apo and Protease

Table 1— Table showing relationship between migration distance in cm of the protein standard
and molecular weight of protein in kDa.
 Molecular Weight (in kDa) Distance Migrated (in cm)
2 9.48
5 8.43
10 6.70
15 5.21
20 4.15
25 3.68
37 2.44
50 1.89

Figure 2— Graph illustrating the distance migrated for the proteins in the protein standard lane
(x-axis) vs. the molecular weight in kDa of each protein (y-axis).

y = -0.1665x + 2.0165
Molecular Weight Standard Curve for SDS-PAGE R² = 0.974
1.8
1.6
Log(Molecular Weight) in kDa

1.4
1.2
1

0.8
0.6
0.4
0.2
0
0 1 2 3 4 5 6 7 8 9 10

Table 2- Molecular weight of myoglobin samples

Using the standard curve and the line equation derived from the graph above (y= -0.1665x +

2.0165), the molecular weights below were found:

 Apparent Molecular Weight in kDa
Apo & Protease 16.81221751
Apo 15.35596098
Holo 16.57513216
Holo & Protease 16.26155229
Apo & Protease Fragment #1 7.632731759
Apo & Protease Fragment #2 5.412152184
Apo Fragment #1 6.069319498

3. The calculated Molecular Weight of Myoglobin:

The molecular-weight of Apomyoglobin and Holomyoglobin with and without protease

is within the range of 15.3 kDa and 16.8 kDa, and the calculated molecular weight of

myoglobin is 16.7, which is within this range and thus matches my experimental results.

4. Question 4:

Based on the structural studies of apomyoglobin, the removal of a heme group will lead

the F helix to be broken into two pieces because it only has one lysine/arginine amino

acid that is not followed by a proline amino acid which is why trypsin can only break the

F helix at one specific site. Thus, we should have 2 bands in the apomyoglobin and

protease sample and the other three lanes should be intact because holomyoglobin (lanes

1 and 2) still has a heme (and thus won’t be affected by the trypsin) and the

apomyoglobin sample (control sample in lane 3) should also be intact because it has not

come in contact with the protease. Unfortunately, our results do not support this

prediction because we have 2 fragments in the apomyoglobin sample (lane 3) and 1

fragment in the apomyoglobin and protease sample (lane 4). This inconsistency may have

been caused due to contamination of lane 3 and 4. We made an error while transferring
 the holomyoglobin, apomyoglobin, and the protease from the tubes into the side walls of

the gel plate. This resulted in some of the protease that was supposed to be in lane 4 to

interact with the apomyoglobin sample in lane 3 and thus create two bands but since

some protease was still left in the apomyoglobin and protease sample we see the

formation of one band in lane 4. As a result, the bands in lane 3 are not in the correct

position and the 2 band that was supposed to form in lane 4 is not visible because there is

not enough protease in lane 4 (the protease was “shared” between lane 3 and 4 and this

resulted in the incorrect formation of the bands).

Note: Right after we started doing the electrophoresis step, Dr. Thieringer noticed that we

did not have enough electrophoresis running buffer, which is why we did not see any

bands form in the first ten minutes of the experiment. This may have also caused our

results to be incorrect.

5. Question 5:

The student may have forgotten to add Coomassie Blue dye, which is the most common

reagent used to stain proteins and help visualize the bands being formed. Thus, it is not a

matter of whether or not the samples were digested by trypsin.

I have completed this lab report in accordance with University regulations and the Honor Code.

X______________________________________