Electrophoresis Separation Of Proteins Cytochrome C, Myoglobin, Hemoglobin, And Serum Albumin By Using Isoelectric Focusing System (Ief) Essay, Research Paper

Electrophoresis Separation of Proteins Cytochrome C, Myoglobin, Hemoglobin, and Serum Albumin by Using Isoelectric Focusing System (IEF)

Introduction

Proteins are composed of amino acids. All amino acids are amphoteric molecules consisting of three types of amino acids: neutral, acidic, and basic. Thus, for any protein there is a characteristic pH, called the isoelectric point (pI), at which the protein has no net charge and therefore will not move in the electric field. Electrophoresis takes advantage of this characteristic. Proteins are electrophoreased, and the most negatively charged protein moves closest to the cathode, and the most positively charged protein moves closest to the anode. Cytochrome C was expected to move closest to the cathode, and serum albumin was expected to move closest to the anode. Only cytochrome C was expected to move to the cathode. The other three proteins were expected to move toward anode. The purpose of electrophoresis was to see how a difference in pI makes a difference in the electrophoretic mobility of protein.

Materials and Methods

Four proteins were electrophoreased by using the Tris-Glysin buffer of pH 8.6 and a horizontal agarose gel 1.1 % in isoelectric focusing (IEF) at a voltage of 175 V and at a current of 79 mA. The agarose gel was made by mixing 0.18g of agarose in 1.5ml of Tris-Glysin buffer with a pH of 8.6. That is 100 % * 0.18 / (0.18 + 15) = 1.1% of agarose gel. 15 ??l of each protein sample was loaded into each sample application well on the agarose gel without mixing with glycerol solution. After the agarose gels were placed on the stage of the electrophoresis chamber, Tris-Glysin buffer of pH 8.6 was filled in the electrophoresis chamber carefully until the agarose gels were slightly covered with the buffer. Four proteins had electrophoreased for about 50 minutes. The agarose gels were removed from the electrophoresis chamber and stained overnight with the Coomassie Blue to visualize proteins in the agarose gel.

Results

Well 1 Cytochrome C pI 10.2

Well 2 Myoglobin pI 7.2

Well 3 Hemoglobin pI 6.8

Well4 Serum albumin pI 4.8

Sample Volume 15 ??l

PH of buffer 8.6

Voltage 175 V

Current 79 mA

Running Time 0.8 hr

Table 1 shows the conditions of this IEF electrophoresis. 15 ??l of each cytochrome C, myoglobin, hemoglobin, and serum albumin were loaded into the well as indicated in Table 1. Well 1 is the bottom well in Figure 1. A voltage of 175 V and a current 79 mA was applied in the buffer of pH 8.6 for 50 minutes, and bubbles were observed on the electrode during the electrophoresis. Figure 1 shows that cytochrome (*1 in Figure 1) moved closest to the cathode, and serum albumin (*4) moved closest to the anode. Myoglobin (*2) and hemoglobin (*3) moved toward the anode, but hemoglobin moved farther from the well than myoglobin.

Discussion

The results support the original hypothesis that cytochrome C will move closest to the cathode, and myoglobin and hemoglobin will move to the anode with serum albumin being the closest to the anode. These results clearly show the relationship between movement of proteins and their isoelectric point (pI). The greater the difference is between pI of proteins and pH of the buffer, the farther the proteins are from the well in this experiment.

The protein with a higher pI than the pH of the buffer was positively charged because it accepted hydrogen ions from the buffer. Then this positively charged protein moved to the negative region, or cathode because it was attracted by hydroxyl ions formed at the cathode by the electrode reactions. When this protein bonded with hydroxyl ions, it became neutral and stopped its movement. On the other hand, the protein with lower pI than the pH of the buffer was attracted by the positive region, or anode, where hydrogen ions were formed. Since this protein released hydrogen ions to the buffer, it became negatively charged and moved to the anode to bond with hydrogen ions to become neutral.

The IEF electrophoresis using agarose gels have been used by researchers, and this technique has proved to be an efficient method for separating small quantities of proteins. U. Ravnskov (1975) states in his article Low molecular weight proteinuria in association with paroxysmal myoglobinuria that agarose gel electrophoresis is a great method to separate myoglobin and hemoglobin. The difference between hemoglobin and myoglobin in pI is 0.4, yet the IEF horizontal agarose gel electrophoresis with 15 ??l of quantity visualized this difference. A study performed by C. Caudie, O. Allauzen, J. Bancel, and R. Later (2000) used agarose gel IEF and IgG immunorevelation to detect IgG oligoclonal bands (OCB). Their conclusion was that IEF with immune detection is the most sensitive and specific test for the detection of chronic CNS inflammation. Similar research was performed by J. Lunding, R. Midgard, and CA. Vedeler (2000) who compared the superiority of IEF, agarose gel electrophoresis (AGE), and IgG index in sensitiveness and specificity in detecting nervous system disorder. Restricted OCB were found in IEF and AGE, and the researchers found that more accurate results were obtained from IEF. Also, IEF was far better than IgG index in determining intrathecal IgG synthesis. As researchers recommended IEF, the migration of all four proteins were successful with IEF using the horizontal agarose gel even with the small amount of protein samples. This technique could be used in analysis, purification, and detection of proteins.

Improvements could be made in the resolution of the protein band in the agarose gel and experimental time. Improvement in resolution could be achieved by reducing the diffusion. An increase of the viscosity of the agarose gel reduces the diffusion, and resolution would therefore increase. The adverse effect of this method is that it would slow down the experiment because the increase of viscosity of the agarose gel increases the friction of proteins. Increasing the experimental time reduces the resolution and thus is not always a successful method to improve resolution. This method would not be a good method for the proteins cytochrome C, myoglobin, hemoglobin, and serum albumin because Figures 2, 3, 4, and 5 show that hemoglobin and serum albumin are greater in size. That is, hemoglobin and serum albumin tend to be influenced by the friction. Another method to improve the resolution is to increase the strength of the electric field. This method also reduces the time of the migration of the proteins. The only thing to be careful with about this method is the temperature of the agarose gel because the high electric field produces heat, and this might cause the proteins to be denatured.

Literature Cited

Ravnskov, U. (1975, February). Low molecular weight proteinuria in association with paroxysmal myoglobinuria. [Abstract] Clin Nephrol 1975 Feb;3(2). 65-9. Retrieved January 31, 2001 from the WWW: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=47277&dopt=Abstract

Caudie, C., Allauzen, O., Bancel, J., and Later, R. (2000 March-April). Role of isoelectric focusing of cerebrospinal fluid immunoglobulin G in the early biological assessment of multiple sclerosis. [Abstract] Annales de Biologie Clinique. Vol. 58, Issue 2, 187-93. Retrieved February 2, 2001 from the WWW: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10760705&dopt=Abstract

Lunding, J., Midgard, R., and Vedeler, CA. (2000 Nov). Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index. [Abstract] Acta Neurol Scand 2000 Nov;102(5). 322-5. Retrieved February 2, 2001 from the WWW: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11083510&dopt=Abstract

Natural Toxins Research Center at Texas A&M University. Isoelectric focusing. Retrieved January 29, 2001 from the WWW: http://ntri.tamuk.edu/if/if.html

Bibliography

Literature Cited

Ravnskov, U. (1975, February). Low molecular weight proteinuria in association with paroxysmal myoglobinuria. [Abstract] Clin Nephrol 1975 Feb;3(2). 65-9. Retrieved January 31, 2001 from the WWW: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=47277&dopt=Abstract

Caudie, C., Allauzen, O., Bancel, J., and Later, R. (2000 March-April). Role of isoelectric focusing of cerebrospinal fluid immunoglobulin G in the early biological assessment of multiple sclerosis. [Abstract] Annales de Biologie Clinique. Vol. 58, Issue 2, 187-93. Retrieved February 2, 2001 from the WWW: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=10760705&dopt=Abstract

Lunding, J., Midgard, R., and Vedeler, CA. (2000 Nov). Oligoclonal bands in cerebrospinal fluid: a comparative study of isoelectric focusing, agarose gel electrophoresis and IgG index. [Abstract] Acta Neurol Scand 2000 Nov;102(5). 322-5. Retrieved February 2, 2001 from the WWW: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=11083510&dopt=Abstract

Natural Toxins Research Center at Texas A&M University. Isoelectric focusing. Retrieved January 29, 2001 from the WWW: http://ntri.tamuk.edu/if/if.html