Laboratory 6:

Determination of protein molecular weight

Gel Filtration Chromatography

It is essential that the biochemist obtain detailed knowledge of the physical properties of a protein to fully understand its function within the cell. Physical properties such as charge, size, amino acid sequence, and higher order structure (secondary, tertiary, and quaternary structure) can be used to identify the protein, design purification protocols, and assay for its enzymatic or structural activities. A particulary useful property of all proteins is their size or molecular weight. Different cellular proteins are most easily distinquished by their differences in size.

The size of a protein can be expressed in two equivalent terms. The term "molecular weight" (Mr, relative molecular mass) is based on a ratio of the mass of a molecule to 1/12 of the mass of carbon 12. The value of Mr is unitless because it derives from the ratio of two masses. The second term, molecular mass, is functionally equivalent to molecular weight, but it refers to the atomic mass of the protein. It is not a ratio and is expressed in the units called daltons (Da). For this exercise, we will use the term molecular weight.

The two most common and widely used methods for protein molecular weight determination are gel filtration chromatography and sodium dodecylsulfate-polyacrylamide gel electrophoresis. Although the relationship between these two methods is not obvious in operation, both rely on the movement of proteins through a porous matrix to distinguish proteins of different sizes. Both methods will give an estimated or apparent molecular weight for a protein. The actual molecular weight of a protein can only be deduced from its primary sequence by adding up the masses of its component amino acids. The estimated and actual Mr for a protein can differ depending on the shape and physical properties of the molecule. In this laboratory exercise you will learn the principle and practice of both of these methods, and apply them to the determination of the molecular weight of a protein.

Gel filtration chromatography (also called molecular sieve or gel exclusion chromatography) is a form of column chromatography. Column chromatography consists of a solid stationary phase and a liquid mobile phase. The stationary phase is confined to a column (glass tube) and the mobile phase (a buffer or solvent) is allowed to flow through the solid phase in the column (see figure 1). A mobile phase containing a dissolved protein or protein mixture will interact with the stationary phase as it moves through the column. The degree of interaction between the stationary phase and the proteins will depend on the properties of the protein, the stationary phase, and the composition of the mobile phase.

Figure 1. Schematic representation of separation by gel filtration chromatography.

Gel filtration chromatography takes advantage of the physical property of molecular size to achieve separation; that is, proteins interact with the stationary phase based on their molecular weight. The general term to describe the substance that makes up the stationary phase is called the gel matrix. In gel filtration chromatography, the matrix is composed of tiny beads (0.1 mm diameter) made from highly cross-linked polymers. The beads swell in the presence of solvent to form microscopic porous sponges. In our case, the solvent will be a buffer. The hydrated beads are packed into a column to form the matrix for chromatography. The porous nature of the hydrated beads, called the gel, forms the basis for the separation method.

Figure 1 illustrates the principle of gel filtration chromatography. A sample containing a mixture of proteins of different sizes (e.g. a cell extract) is introduced to the top of the gel and allowed to flow into the matrix. Buffer is continually added to the top of the column as the mobile phase moves down through the matrix. As the mobile phase exits the bottom of the column, it is collected as aliquots of constant volume into a series of test tubes. The exiting mobile phase is called the column eluate, and each successive portion of the eluate is called a column fraction. The total volume of the column occupied by the gel matrix and the mobile phase within and between the beads is called the total bed volume (Vt). The matrix that is packed into the column is often referred to as the column bed.

As a mixture of proteins flows through the column, proteins or molecules larger than the largest pores of the gel cannot enter the pores (they are excluded) and they pass rapidly through the column between the beads with the mobile phase. The volume of the column bed that is excluded from the pores of the matrix is called the exclusion volume or void volume (Vo). Large proteins that do not enter the pores will elute from the column in a volume equivalent to the void volume. Smaller proteins can enter and exit the pores of the gel by diffusion (they are included). Therefore, their movement down through the column will be retarded and they will require more time to elute. The volume of the mobile phase that is required to elute a particular protein is called its elution volume (Ve). The porosity of the beads is an important feature in gel filtration because it determines the size range of proteins that can be separated by the method. Different matrices are available commercially that differ in the extent of cross-linking and therefore, their pore size. Beads with large pore sizes are more effective at separating large proteins.

The volume that is included in the pores of the matrix is called the inclusion volume or internal volume (Vi). The volume occupied by the polymer in the beads is called the polymer volume (Vp). The packed bed of a porous matrix can be represented mathematically as

Vt = Vo + Vi + Vp.

The molecular weight of a protein can be estimated by comparing its elution profile with the elution patterns of standard proteins of known molecular weight. A linear relationship is obtained if the logarithms of the molecular weights of standard proteins are plotted against their respective elution volumes. The molecular weight of the unknown protein can be estimated by extrapolating from the standard graph.

Gel filtration chromatography will give you the apparent molecular weight of the native protein. The proteins do not undergo any harsh treatments prior to gel filtration chromatography. Therefore, they maintain their secondary, tertiary, and quaternary structure. This property of the method makes it very valuable for characterizing and purifying proteins whose functional properties (i.e. enzymatic activity) will be studied in subsequent experiments.

Experimental procedures I

In this exercise, you will determine the molecular weight of myoglobin by comparing its elution volume from a gel filtration column to those of a standard protein and a dye. All of the molecules in the standards and the myoglobin are colored. This makes the analysis of the elution profile convenient. A list of the molecules and their characteristics are given in table 1.

Table 1.

Myoglobin - Myoglobin is composed of a single polypeptide chain that is bound to a single prosthetic group called heme. The iron in the heme group functions in binding oxygen. Myoglobin is found in muscle where it binds and stores oxygen for use during respiration by muscle cells. The iron-heme complex gives myoglobin its characteristic color.

Hemoglobin - Hemoglobin is the major protein found in red blood cells (erythrocytes). It also contains an iron-heme group that binds oxygen. This protein serves to transport oxygen from the lungs to peripheral tissues. Hemoglobin is a tetramer (i.e. it contains four polypeptide subunits) that associate by non-covalent interactions. The subunits are called alpha and beta and are of nearly identical molecular weight. The color of hemoglobin depends on whether it contains bound oxygen, but it usually is red to red/brown.

Phenol red - Phenol red is a small dye that is used as a low molecular standard in gel filtration chromatography.

Blue Dextran - Blue Dextran is a large polysaccaride containing a covalently attached blue dye. It is used as a size standard in numerous biochemical exercises. You will use it to determine the exclusion volume of your column.

Column flow rate

1. The chromatographic column with its components is shown in figure 2. The column already contains the gel filtration matrix (white gel) that has been equilibrated in the mobile phase (phosphate buffered saline or PBS, see laboratory 2).

Figure 2. Column set-up for gel filtration chromatography.

2. Buffer should flow through your column at 8-10 ml/hr. Fill the column reservoir with buffer by slowly pipetting PBS onto the top of the gel matrix until the buffer is 5 cm above the gel surface. Be very careful not to disturb the surface of the gel or the resolution of your chromatography will be effected. Finish filling the reservoir.

3. Place a 10 ml graduated cylinder under the column to catch the buffer that will flow through.

4. Remove the blue cap from the column and allow the buffer to collect in the cylinder for exactly 10 minutes. Replace the blue cap to stop the flow.

5. Measure the approximate volume of the column eluate in the cylinder. Record this value and calculate the flow rate. Enter these values in your notebook as given below.

FLOW RATE

_______________ ml/10 min

_______________ ml/hour

6. Start the column flow again by removing the blue cap. Collect 5 drops in a small test tube and stop the column by replacing the cap. Measure the volume of the 5 drops you collected. From this volume, calculate the number of drops that will give 350-400 ml. This will be the volume of each of the fractions that you will collect in the following section. Note the exact volume per drop. This will be necessary to calculate the elution volumes in the next section.

Separation of the protein and dye mixture

1. Each group will be given a solution containing a mixture of the colored proteins and dyes described above.

2. Obtain 25 small test tubes to collect your eluate fractions. Number them 1-25.

3. Remove the excess buffer from the column reservoir with a Pastuer pipet.

4. Remove the blue cap from the bottom of the column and allow the buffer to drain down to the level of the gel bed. Replace the blue cap to stop the flow.

NOTE: Do not allow the buffer to fall below the surface of the gel bed or your column will go dry and be useless. Therefore, you must pay close attention to the level of the buffer as it approaches the bed surface.

5. Transfer 100 ml of the protein-dye mixture to the top of column bed using a P200 pipettor. Place the tip of the pipet about 1-2 mm above the surface of the gel bed. Slowly and carefully layer the sample onto the upper bed surface without disturbing the surface of the gel.

6. Remove the blue cap and allow the mixture to enter the bed. Stop the flow and add 200 ml of PBS to the column. Allow this to flow into the column. Stop the flow by placing the blue cap on the column.

7. Fill the column with buffer. Carefully fill the reservoir with buffer.

8. Place tube #1 under the column exit and resume the flow of the column. Begin to collect the first column fraction. Collect the appropriate number of drops to give 350-400 ml (calculated above) in tube 1.

During the chromatography, periodically add buffer to the column reservoir to keep the level of buffer constant. This will insure that the flow rate through the column is constant and the volumes of all your fractions are equal.

9. After the appropriate number of drops are collected in tube 1, replace tube #1 with tube #2 and collect the second fraction.

10. Continue collecting fractions, switching to a new tube after collecting the appropriate number of drops until all of the colored substances have been eluted from the column. Approximately 20-25 fractions should be collected. You will notice that the colored substances begin to separate from one another as they flow through the column.

11. Examine each of the column fractions and determine which fractions contain the highest concentrations of each of the substances listed in Table 1. This is most easily accomplished by holding the fractions up against a sheet of white paper. The color of the wells will be your guide to the identification of each protein.

NOTE: The molecules elute in the order blue dextran, hemoglobin, myoglobin, phenol red.

NOTE: The colors of hemoglobin and myoglobin are very similar. It may be difficult to distinguish their elution peaks. Ask your teaching assistant for advice.

12. Record the fraction numbers and elution volumes for each substance in your notebook using a table with headings similar to the one below.

13. Calculate the Vo of the column using blue dextran.

14. Plot the Ve vs. log Mr using graph paper for the hemoglobin and phenol red. Do not include the Ve for blue dextran because it elutes in the void volume.

15. Extrapolate the Mr of myoglobin from your graph.