A Study on Proteins, Enzymes, & Catalytic Activity
Basis in Column Chromatography, Enzyme Activity Assay, Protein Assay, Gel Filtration, and Enzyme Assay
Proteins are the building blocks of our bodies and many fall into the category of enzymes which play critical roles in the biochemical reactions which keep us alive. Understanding enzymes, and proteins in general, is crucial to our overall understanding of the biochemical make-up of our internal systems, as well as those of Earth’s other organisms. Horseradish peroxidase (HrP) is an example of an enzyme that is sometimes utilized in biochemistry for its economic benefits and ease of isolation. In this experiment, column and gel-filtration chromatography were used to isolate HrP. Enzyme assays were then used to determine the catalytic activity of the isolate. Determination of the molecular weight was made through gel electrophoresis. These techniques were shown to be viable and are crucial for proper protein analysis, and yielded a better understanding of HrP’s catalytic activity.
Proteins are types of macromolecules consisting of chains of amino acid residues.
Protein production begins with transcription of mRNA within the nucleus and ends within the confines of the rough endoplasmic reticulum and golgi apparatus. Once transcription is complete and the spliced mRNA is chaperoned out of the nucleus through the nuclear pores, ribosomal subunits come together and begin translation of the mRNA, beginning at the start codon and reading along until terminated. Every three nucleotides forms a codon which within the ribosome allows the attraction and chemical reactions required to develop a polypeptide. Amino acid residues make each unit of the polypeptide up, linked
via their peptide bonds, and they can be substantially large for some proteins (Voet et al., 2013). Once translation is complete the protein must be properly folded into its correct shape so that it may function properly and meet the cell’s needs. Often the proteins created within the cell are enzymes, or proteins which catalyze chemical reactions (Klug et al., 2009). Enzymes drive the metabolic process and without the work they do, lowering activation energy required for reactions to progress, we would not be able to function.
Proteins typically have very specific functions in the body. For instance the two proteins, Myoglobin and Hemoglobin, both bind oxygen, but they have vastly different roles. Myoglobin is found within the tissues of the muscles, binding oxygen for muscle cells. It contains an iron-heme pigment group which gives our muscles their red coloration (Voet et al., 2013). Interestingly, myoglobin was the first protein to be three-dimensionally determined using X-ray crystallography, achieved by John Kendrew and associates in 1958 (NSF, 2004). Hemoglobin, in contrast to myoglobin is much more complex, essentially containing four myoglobin-like subunits, two α-globin and two β-globin, thereby allowing one hemoglobin to carry four oxygen molecules. (Alberts et al., 2008) hemoglobin is responsible for carrying oxygen from the lungs to the peripheral tissues for almost all vertebrates.
In this experiment we look at horseradish peroxidase and myoglobin specifically. HrP also contains a heme pigment group with an Iron atom like myoglobin and hemoglobin. Therefore it is a metalloenzyme as well, on top of being a glycoprotein (Collins, 2014). The heme group cofactor seen in these enzymes is depicted by figure 1, showing the binding of a molecule of oxygen and some of the resonance which occurs to facilitate this binding.
Horseradish peroxidase is found in the roots of horseradish plants (Armoracia rusticana). Its overall structure is mostly alpha helical as depicted by figure 2 (Constantino et al., 2010), with some irregular loops and the heme cofactor at its center.
Myoglobin, as stated previously is a monomeric enzyme which contains a “heme prosthetic group” (Voet et al., 2013) and falls into the globin family of proteins. The ring system which makes up heme is heterocyclic in structure, and contains four pyrrole groups with methene linkages. Myoglobin’s iron oxidizes to Fe(III) from Fe(II) forming metmyoglobin or methemoglobin, which gives old meat and dried blood their brownish colorings. Just like in hemoglobin, other small molecules beside oxygen can bind to myoglobin’s heme rings, such as carbon monoxide, nitrogen oxide, and dihydrogen sulfide, many of which have a greater affinity for the binding site then oxygen. This causes the active site which makes this enzyme useful to become blocked. (Voet et al., 2013)
Proteins in general can be purified or isolated using multiple techniques such as centrifugation, electrophoresis, as well as a multitude of different chromatography techniques. Other tests typically conducted on proteins for determination of activity and structure include x-ray crystallography, nuclear magnetic resonance, and mass spectrometry, to name a few.
Materials & Methods
The procedure and materials used were in following with the published procedure (Collins, 2014).
Kinetic data plotted for determination of enzyme activity in absorbency units/time. Table 1 refers to all data obtained via each fraction from the affinity column which was eluted and read. Figure 3 depicts this data showing the slope rate vs fraction number, in essence the slope of the absorbency units/time, in this case giving a visual depiction of enzyme activity.
Figure 4 shows a chromatography profile graph derived from the affinity column data in the fractional table. Both the readings at 280 & 403nm were plotted. Enzyme activity in absorbance units over time was plotted against the fraction number. Table 2 depicts the calculated data for the crude, pellet, supernatant, and pooled fractions samples. Each sample volume was a ten microliter sample which was converted from the slope/kinetic data into the enzyme activity/sec/mL.
The graph below depicts the enzyme assays by fraction against their absorbance. This is a linear plot depicting enzyme activity. Fractions 12 and 16 are not shown because they did not have any activity in their samples.
Cary 300 UV/VIS absorbance fraction graph for the fraction sets in the UV/VIS spectrometer. Fractions show relative absorbance for each sample measured.
Gel Filtration Columns
This graph, Figure 7, depicts the log M.W. of standards vs their Kd values, using this standard it is possible to evaluate myoglobin and peroxidases molecular weight. Myoglobin is approximately 17 Kda, while Peroxidase is approximately 44 Kda (main peptide chain minus sugar and calcium/heme is 34Kda.
This graph, Figure 8, depicts the BSA standard ran at 595nm on the Milton Roy UV/VIS spectrometer.
The following graphs, Figure 9 and 10, depict the Gel Filtration Column data for Peroxidase and Myoglobin.
Here, Table 3 depicts the electrophoresis protein standards, giving the relative mobility (Rm) for each protein sample that was ran in a lane. Figure 11 is a graph showing the log molecular weight versus the Rm.
Table 4 gives the molecular masses of each protein as determined by gel filtration, SDS, and the current scientific literature.
This experiment highlighted multiple lab techniques crucial to working with proteins. Enzyme activity and purity were determined following gel filtration. For the affinity chromatography, concanavalin-A, a lectin, was used in the column as it binds to peroxidase readily via binding to the carbohydrate side chains (since it’s a glycoprotein), which allows everything else to flush through the column. Once the rest is through, the use of mannose, which concanavalin-A has a higher affinity for, is utilized to wash the peroxidase back into the matrix for collection. Another type of lectin, such as lentil lectin or snowdrop lectin could be used, which also have an affinity for mannose.
The fold purification seen in table 2 shows how much the affinity column purified the sample, where the crude is at 1, since the samples specific activity is divided by its own specific activity. This is because this sample contains all the peroxidase possible. The supernate had a fold of 10.6, the re-suspended pellet had a fold of 2.0, and the pool(13+14) had a fold of 10.8, slightly higher than the supernate. The supernate here should indeed have a higher specific activity and if the gel column does well, higher fold. This is seen, and should also be expected in the pool as the pool was selected to highlight the most probable elutions with the highest percentage of peroxidase. The pellet after being re-suspended following the pour off of the supernate should contain little peroxidase, yielding a lower specific activity and a lower fold, which is what is observed. Yields for the supernatant and pool were high, with crude being at 100% as expected. The pellet showed a lower yield which is also expected. Yields over 100% shouldn’t be observed but this could be attributed for high concentrations of peroxidase in low volume solutions and improper calculations attributed to mis-use of proper units.
The molecular weight of myoglobin in the literature is 17Kda, with peroxidase molecular weight being (total) 44Kda. The peroxidase peptide chain alone is ~34Kda. The 44Kda literature value includes the peptide chains, heme, calcium, and carbohydrate groups. Gel filtration, allowed using the Kd vs log of m.w. showed lower Myoglobin, which was also seen in the SDS-PAGE from the electrophoresis with a m.w. of 6.3Kda, which is substantially lower than the lit value of 17.0Kda. This could be due to discrepancies during the development of the myoglobin solution or there could actually be two, hard to distinguish bands in the same area, which would give about 14Kda which is closer to the lit value. Peroxidase did well, with experimental values of 57Kda total from the SDS-PAGE, which can most likely be attributed to additives and additional residues left over in the solution, which can be seen to still be associated with the pellet assay. Subtracting these out would yield a more reasonable number around the literature value.
Visually, the enzymes obtained were not very pure. The dying that occurred was very hard to discern immediately following removal of the gel from the dye solution. The yield % therefore is incorrect, but the fold calculation is more discernible and shows the overall purity of the sample well. This estimation is reasonable in the sense that if there is little enzyme in the wells then there will be less enzyme for the dyes to adhere to, which correlates to an image that is harder to work with. That being said, the peroxidase was most likely not pure for the fact that the molecular weight data also showed it having a higher overall Kda than the literature values. Reasonably, this can be assigned to impurities in the sample.
- Collins, J., Proteins and Catalytic Activity,Biochemistry I Laboratory Experiments [Online] 2014, 13th edition. Blackboard. http://blackboard.usi.edu/ (November 24, 2014).
- Voet, Donald., Voet, J. G., Pratt, C. W., Fundamentals of Biochemistry, 4th 2013. p93-354.
- NSF, (U.S.) National Science Foundation: Protein Data Bank Chronology [Online], 2004. http://www.nsf.gov/news/news_summ.jsp?cntn_id=100689 (Jan. 21, 2004).
- Constantino, Z., Amedeo, P., Andrea, A. “On the catalytic role of structural fluctuations in enzyme reactions: computational evidence on the formation of compound 0 in horseradish peroxidase”. Faraday Discuss, 2010. 145,107-119, DOI: 10.1039
- Klug, W., Cummings, M., Spencer, C., Palladino, M., Concepts of Genetics, 9th 2009. p6-9.
- Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K., Waleter, P., Molecular Biology of the Cell, 5th 2008. p124-193.
acrylamide protein gel electrophoresis
enzyme catalytic assay
polyacrylamide gel set-up 02
affinity column, fractions
polyacrylamide gel set-up 01
Date: 24 November 2014