Experiment 7: Electrophoresis of Proteins

Alex Foo (Partner: Matthew Crerar)

5673657 – 7acyf@queensu.ca

Bchm 317 – Group 3

TA: Da Duan
November 10, 2009
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Introduction

Electrophoresis is one of the most versatile tools in analytical biochemistry, as it

allows separation of proteins and other molecules based on a number of different

properties. However, in order to use electrophoresis effectively in the laboratory, one must

have a thorough understanding of the mechanism of separation for different

electrophoresis methods, and the strengths and limitations of associated with each

method. In this study, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-

PAGE) is used to separate proteins from samples of purified invertase, and to determine

the molecular mass of rat Lactate Dehydrogenase (LDH) isozymes. Cellulose Acetate

Electrophoresis (CAE) is then used to resolve the different LDH isozymes. The properties

and limitations of both techniques will be explored during the course of the study.

Materials and Methods:

All materials and methods used in this laboratory experiment are found in the Queens

University Bchm 317 lab manual [1], and were used without modification.
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Results: *Note: all tables and figures are shown in

the appendix

In this experiment, SDS-PAGE gel electrophoresis was used to determine the

molecular weight of Lactate Dehydrogenase Isozymes (LDH) from rat heart and muscle

tissue. The molecular weight of carboxypeptidase was also measured. Molecular weight

measurements were calculated by comparing the distance each of the sample bands

migrated to a standard lane loaded with peptides of known molecular weights, as shown in

figure 1. The 4 lysosome purification fractions from previous experiments were also run on

the same gel to qualitatively observe the purity of the various fractions. This is done by

both observing the size and intensity of the invertase protein band, and the number and

intensity of other contaminating protein bands on the gel.

The distance migrate and mass of each of the standard proteins was tabulated in

table 1, and the data was used to create a standard curve, as shown in figure 2. The

standard curve was used to calculate the molecular weight of the two LDH isozymes, as

well as the carboxypeptidase. Sample calculations are shown in calculation 1, and the final

molecular weights are tabulated in table 1.

Since the molecular weight of invertase subunits is about 60-63 kDa depending on

the isozyme studied [2][3]*, it is expected that invertase will have a migrate about 0.81

cm to form the pair of closely spaced bands (one for each isozyme) as indicated in figure

1. It is apparent that the intensity of the invertase band is highest for fraction 4. Fraction 2

was also observed to have a high invertase band intensity, which contradicts previous

studies which indicate that fraction 2 has the lowest invertase activity of all the fractions.

The intensity and number of the contaminating protein bands in fraction 2 suggests that,

although fraction 2 may have a high invertase content, it also has a lot of contaminating

*In addition to searching literature values, the “theoretical” molecular weight of invertase was obtained as follows. The amino acid
sequence for both invertase isozymes from Saccharomyces cerevisiae were obtained from the Swissprot database, and used to
calculate their molecular weights using the Protparam tool from the ExPASy Proteomics Server, Swiss Institute of Bioinformatics.
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proteins, and thus its total purity is still low. This matches with previous observations

showing that fraction 2 has the lowest specific activity, but still does not explain the

anomalous result for absolute invertase content. Fractions 1 and 3 were observed to have

lower invertase band intensities, with fraction 3 showing fewer contaminating protein

bands than fractions 2 and 1. This suggests that fraction 3 has both a greater absolute

invertase content and invertase purity than fraction 1, which correlates to results from our

previous study. A summary of the qualitative observations from the SDS-PAGE of the

invertase fractions, and the quantitative results from our previous study are shown in

table 2.

It was noted that the SDS-PAGE procedure did not produce any significant

separation of the two LDH isozymes. For this reason, it was decided to examine LDH

isozymes using cellulose acetate electrophoresis instead of SDS-PAGE in order to achieve

better separation. Samples of LDH from rat heart, kidney and liver tissues were placed on

separate cellulose acetate strips. Voltage was then applied lengthwise across the strip to

separate the different isoforms. The strips were then stained using an “activity stain” to

help visualize the position of the various bands.

It was noted that this method gave a much better separation pattern compared to

the SDS-PAGE, with the liver LDH sample producing two bands – one band occurred about

halfway down the strip (position 1), while the second band occurred close to the starting

position (position 2). This corresponds to the presence of a strongly negatively charged

isozyme, and a less charged isozyme. The kidney LDA sample produced only one band

which roughly corresponds to the halfway band of the liver extract (position 1), which

suggests that the kidney sample only possesses the strongly chargedisozyme. The heart

LDA extract showed a band close to position 1 (position 3), and a band at position 2. It also
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showed two additional bands bracketing position 2, thereby indicating that the heart

sample contains a large number of LDA isozymes, each with a different degree of charge.

The cellulose electrophoresis results are shown in figure 3, alongside the labels for

position 1, position 2 etc. This separation pattern is much more useful than the single

band at ~60 kDa obtained through SDS-PAGE.

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Discussion:

In this laboratory experiment, we initially employed SDS-PAGE as a means for

protein separation. The basic principle behind gel electrophoresis is that charged

molecules such as peptides will migrate through a supporting medium due to an

electrostatic force, brought about by the application of an electric field across the gel. The

speed of migration will depend on the equilibrium between the electrostatic force exerted

by the electric field, and the opposing frictional and electrophoretic retardation forces, as

shown in figure 4. SDS is added to the gel in order to give all proteins the same linear,

unfolded structure and charge to weight ratio. This ensures that they all have the same

electrostatic force to mass ratio, and that separation occurs based upon the size sensitive

electrophoretic retardation force, thereby allowsing separation based on peptide size

alone. It should be noted that, as a result of the charge/weight ratio homogenization from

the addition of SDS, it is impossible to determine the native state of the sample proteins

based on SDS-PAGE gel results alone. However, it is possible to quantify the molecular

weights of the separated proteins through the use of a standard lane on the gel which is

loaded with peptide markers of known molecular weights. By measuring the distance each

of the markers move, it is possible to plot a reproducible, linear relationship between

molecular weight, and distance traveled and use it to determine the exact molecular mass

of the unknown protein samples.

Through the use of SDS gel electrophoresis, we were able to separate the

component proteins from our invertase protein fractions in order to qualitatively observe

their purity. As discussed earlier in the result section, it was found that fraction 4 was the

purest, having the strongest invertase protein band and the smallest amount of

contaminating protein bands. Strong 25 and 37 kDa bands were also observed in fraction
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4, which may be due to the accidental cleavage of invertase into smaller peptide chains

during the course of the purification experiment. It is known that invertase occurs

naturally as a glycoprotein with a mass of 205 kDa [2], of which only 60 kDa make up the

actual invertase. Thus, it is also possible that the lower molecular mass fragments are

simply due to pieces of the glycoprotein shell of invertase breaking off and physisorbing

Coomassie Blue dye from the staining procedure.

The results also showed that fraction 2 showed two strong bands at about 60 kDa.

This suggests that fraction 2 contains a high level of invertase, as discussed previously.

This contradicts our previous study, which showed that fraction 2 has a lower level of

enzyme activity than both fraction 1 and 3 (see table 2). This result may have been due to

the fact that the test tubes were mixed up in the previous study, and thus the calculated

protein concentration of fraction 2 may have been wrong. This could have lead to the

incorrect dilution of the SDS-PAGE protein sample, and thus the abnormally strong

invertase protein band observed fro fraction two. This would also explain why all the bands

in fraction 2 are more intense than any other fraction examined. Another possible

explanation is that there happens to be a second contaminating protein whose molecular

weight also corresponds to 60 kDa.

If one chooses to disregard fraction 2 for the reasons mentioned earlier, and in our

previous report, then the results of the SDS-PAGE separation show that the purity of

invertase increased as the purification process progressed. This is in accordance with the

results observed in the previous lab and previous studies by Neuman and Lampen [4].

Unlike SDS-PAGE, cellulose acetate electrophoresis (CAE) separates proteins and

peptides based on their natural charge instead of their size [1]. Since both LDH isozymes

sampled previously on the SDS-PAGE were observed to have similar sizes, it is expected

that CAE will give better separation. In order to do this, a uniform electric potential is
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applied lengthwise across the cellulose acetate support. This support provides a relatively

constant frictional retardation force upon the proteins while the electrostatic force pushes

proteins through the film at a rate dependant on its natural charge: the more negative the

protein, the stronger the electrostatic force and the faster it will migrate through the

acetate film. Since separation is not based on size, it is impossible to determine the size of

the sample proteins based on the cellulose acetate electrophoresis results. It may be

possible to determine qualitatively the native charge of the sample proteins tested.

However, it is doubtful whether the relationship between charge and distance migrated is

linear or reproducible. Since CAE relies on the proteins’ native charge to function, the use

of SDS should be avoided, as it will eliminate the difference in the proteins’ native charge,

and thus make separation impossible.

In order to visualize the results of the electrophoresis separations, one must have a

method for staining the protein bands on the gels. In the SDS-PAGE study, we were looking

to identify all the protein bands, and thus the Coomassie blue dye was chosen for its

ability to indiscriminately dye all proteins and peptides. In the cellulose acetate study

however, we were only concerned with the LDH peptides, and did not want any interfering

bands from contaminant proteins. Thus, we used an “activity stain” to produce colored

bands only where LDH was present.

The CAE studies show the presence of varying amounts of isozymes in the different

tissues. These different rat isozymes arise due to the fact that LDH is a tetramer in its

natural state. There are two possible subunits, subunit A and subunit B [6], which come

together in different combinations to form the different isozymes observed on our CAE

images. Rat liver tissues produce almost all A subunits, with very few B subunits [6]. This

means that the observed LDH will either contain 4 A subunits or 3 A subunits and one B

subunit [6]. Since the amount of B subunits is very low, the probability of incorporating

two B subunits together in the same LDA molecule is almost zero. This results in the two
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bands observed by both ourselves and [6]. In contract to this, the heart tissue cells

produce almost equal amounts of both subunits, which leads to the presence of 5 different

isozymes [6]. However, the prevalence of one of these isozymes is about 1% [6], which

makes it almost impossible to detect using our CAE method, and thus only 4 isozymes are

observed. The kidney tissue was observed to produce only one band, which suggests that

rat kidney tissue cells produce only one type of subunit, and thus only one isozyme is

possible. However, this was not found to be the case in [6]. This discrepancy could be due

to the fact that cells from different parts of the kidney could express different proteomes,

and thus lead to variability between samples of the same tissue. For example, exercise

and metabolic activity may affect the expression of various isozymes [7], and thus lead to

variability in CAE results. A full comparison between our results and that of from literature

is shown in table 3.

Sources of Error:

As mentioned previously in the discussion, sources of error could have been

propagated up from the previous studies, and have lead to incorrect dilutions of our

invertase fractions, particularly for fraction 2. Other sources of error include the fact that

the expressed proteome for a cell varies with environmental conditions, and even the part

of the tissue from which it was extracted. As discussed in the discussion, this could have

lead to discrepancies between the number of LDH isozymes identified in our study, and

results from literature. Additional sources of error include our inability to guarantee a

uniform electrical fields during electrophoresis, lack of uniformity of our SDS-PAGE

gel/cellulose film, and mistakes in dying and fixing our electrophoresis images.

Conclusion:

In this experiment, we were successful in employing SDS-PAGE electrophoresis to

determine the purity of each of our invertase fractions collected from previous
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experiments. We were also able to determine the molecular weight of two different rat

LDH isozymes. Although these values matched those of literature, they were too close

together to allow proper resolution via SDS-PAGE. As a result, CAE was employed on

samples of LDH from 3 different tissues. This method was able to differentiate between

the different isozymes, and the results roughly corresponded to literature research, with

some exceptions. In using these two techniques, the principles, limitations and possible

applications of various types of electrophoresis were revealed and discussed.

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Sources Cited

1. Prosser, David E., and Vinay K. Singh. (2009) BCHM317 Laboratory Manual 2009-2010.

Queens University, Kingston. pp. 35-37

2. Moreno, S., Sanchez, Y., and Luis Rodriguez. (1990) Purification and Characterization of

the Invertase from Schizosaccharomyces pombe – a Comparative Analysis with the

Invertase from Saccaromyces cerevisiae. Biochem. J. 267. pp. 697-702

3. Trimble, R.B., and Frank Maley. (1997) Subunit Structure of External Invertase from

Saccaromyces cerevisiae. The Journal of Biological Chemistry 252(12). pp 4409-

4412.

4. Neumann, N. P., and J. O. Lampen. (1967) Purification of Yeast Invertase. Biochemistry

6(2). pp 468-475

5. Goodwin, A.B., Schneider, R.E., and William E. Fry. (1995) Use of Cellulose-Acetate

Electrophoresis for Rapid Identification of Allozyme Genotypes of Phytophthora

infestans. Plant Disease 79(11). pp. 1181-1185.

6. Nadal-Ginard, Bernardo. (1978) Regulation of Lactate Dehydrogenase Levels in the

Mouse. The journal of Biological Chemistry. 253(1). pp. 170-177

7. Garbus, J., Highman, B., and Paul D. Altland. (1964) Serum Enzymes and Lactic

Dehydrogenase Isozymes After Exercise and Training in Rats. Am. J. Physiol 207. pp

467-472.
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Appendix:

Figure 1: Stained SDS-PAGE gel after electrophoresis of rat heart and muscle LDH

isozymes, carboxypeptidase, and the various invertase fractions collected from

previous experiments. Lane 4 is the standard lane, with the molecular weights

labeled.

Figure 2: Standard curve for migration distance of the various bands vs the log of the

molecular weight. The linear regression for the linear section of the graph is shown.

Table 1: Tabulated values for the migration distance and molecular weights of the various

proteins run on the SDS-PAGE gel. * denotes values used in the sample calculation

(calculation 1)

Mass
(kDa) Migration (cm)
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250 0.2
150 0.3
100 0.4
75 0.6
50 1.1
Markers
37 1.5
25 2.1
20 2.7
15 3.2
10 3.9
LDH(M4) 35 1.7
LDH(H4) 35 1.7
Carboxypepti
dase 31* 1.9*
Invertase 56 0.9

Calculation 1: Determining the molecular weight of Carboxypeptidase through its

migration distance as observed during SDS-PAGE analysis.

logMW=-0.256X+1.983 X = Distance migrated, X = 1.9cm

logMW=-0.2561.9+1.983

logMW=1.4966

MW=31.376

MW=31kDa
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Figure 3.1, 3.2: Image of the developed CAE films showing the separation of the

different rat tissue LDH isozymes.

Figure 4: Diagram of the various forces acting upon proteins during electrophoretic

separation.
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Table 2: Tabulated qualitative values for invertase sample purity as determined from

figure 1, compared to the invertase sample purity as determined in our previous study. 1

denoted the most pure, and 4 denotes the least pure, as determined by the SDS-PAGE

analysis.

Fracti Specific Activity, U/mg(From Purity (from SDS-PAGE,

on lab 6) Experiment 7)
1 2865 4
2 216.9 2
3 3655 3
4 5039 1
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Table 3: Comparison of the LDH isozymes present in the various rat tissues as

determined by our experiment and literature results [6].

Tissue Experimental values Literature values [6]

5 isozymes present, One of these occurs in trace
Heart 4 isozymes observed amounts only
Kidne 5 isozymes present, all occur in significant
y 1 isozyme observed amounts
Liver 2 isozymes observed 2 isozymes observed