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.
 

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.
 
 Figure 2. Column set-up for gel filtration chromatography.
 
 
http://newarkbioweb.rutgers.edu/bio301s/Lab%204-%20mol%20wt-
%20column%20chromatography.htm