Separating and Sequencing Proteins

WAYS OF SEPARATING MIXTURES OF AMINO ACIDS

n 1. Partitioning chromatography

If a compound is added to a mixture of two immiscible liquids, part will dissolve in one liquid, and part will dissolve in the other. The greater quantity will dissolve in the solvent in which it is most soluble.

A mixture of amino acids is added to a separatory funnel containing two immiscible solvents.

n The more non-polar aa’s will go into the butanol and the polar ones will go into the water.

n Each amino acid has a characteristic partition coefficient which in this case equals [aa in butanol]

[aa in water].

Partitioning chromatography
using columns

n Partitioning can be done using a column of granules which are hydrated with a tightly bound layer of water. Running the mixture of aa’s and n-butanol over the particles partitions the amino acids between the water and the mobile butanol. The amino acids will elute off the column at different rates. The most non-polar ones would dissolve in the butanol most readily and come off first followed by the more polar amino acids.  

2. Ion-exchange chromatography

The basis of this type of separation is difference of charge. The amino acids are separated on columns of plastic beads with sulfonic acid groups, -SO₃H, covalently attached.

The positively charged amino acids will displace the Na⁺ on the beads.

§ The amino acids are added at a pH which is well below the pk’s of most R-groups, but above the pK of the carboxyl group; therefore, the amino acids will be largely positively charged. They will differ in the degree of positive charge. The positively charged amino acids will displace the Na⁺ on the beads. The most positively charged amino acid will bind the tightest such as lys, arg, his. The amino acids are eluted off with a base. The anionic appear first, and the most cationic last.

3. Isoelectric point focusing

n Each amino acid has a different isoelectric point. This difference is an effective characteristic to use as the basis of separation.

A gradient of pH is established in a polyacrylamide gel using ampholytes. These compounds distribute themselves in an electrical field according to their isoelectric points and create the desired gradient of pH, for example pH 2 to pH 12. The mixture of amino acids is then added to the gel and an electric current passed through the gel. The amino acids will migrate to the pH in the gel which is equal to their isoelectric point. The gel can be stained with dyes to locate the bands which represent the different amino acids.

WAYS OF SEPARATING MIXTURES OF PROTEINS

§ 1. Polyacrylamide gel electrophoresis

§ A) Non-denaturing gel

§ A gel is synthesized from acrylamide cross-linked with methylene-bis, acrylamide. The proteins are applied to the gel and an electrical current applied to the gel. The proteins with the greatest amount of native negative charge will migrate toward the anode (+) the fastest. The proteins which are small and compactly shaped move through the pores of the gel more rapidly also.

Non-denaturing gels separate proteins on the basis of differences in size, shape and charge.

§ If the proteins are in the native conformation when loaded on a non-denaturing gel, the proteins will separate on the basis of a combination of differences in size, shape and native charge. Protein bands can be seen when stained with a colored stain such as Coumaisse Brilliant Blue.

§ B. Denaturing gels

§ SDS, sodium lauryl sulfate, an anionic detergent can be added to the sample to give the protein a uniform negative charge. The protein migrates solely according to the size, MW, of the protein.

A graph can be made that plots the log₁₀ MW vs. distance traveled in the gel.

n The MW of an unknown protein can be determined by comparing the distance it travels in the gel to the distances standard proteins of known molecular weight traveled.

C. Isoelectric point focusing

§ Isoelectric point focusing is a type of electrophoresis. The gel in this case contains molecules with different pI’s, called ampholytes. When electrophoresed, these molecules form a pH gradient. The various proteins applied to one of these gels will migrate to a pH equal to their pI. This is a very sensitive method of separation. One unit charge difference is sufficient to separate two proteins.

2. Column chromatography

n Glass columns can be filled with beads of a wide variety of different types.

n Three examples are:

n A) ion exchange resin beads to separate proteins on the basis of charge differences

n B) inert beads of different sizes to allow filtration separation based upon size and shape of the protein

n C) hydrated beads to allow partitioning chromatography based upon differences in solubility.

3. Equilibrium centrifugation

§ In this technique a density gradient is made using a heavy liquid such as a sucrose solution. The sample of proteins is applied to the top of the gradient and the gradient is centrifuged. The centrifugation is stopped before the different protein bands reach the bottom of the gradient. The rate at which a protein moves toward the bottom of the gradient is a function of its size, shape and density. A unit of measure called a Svedberg Unit is used to compare how rapidly different proteins move when the gradient is centrifuged.

SEQUENCING PROTEINS
WHY IS IT USEFUL TO KNOW THE AMINO ACID SEQUENCE OF A PROTEIN?

§ 1. It can be compared to the sequence of other known proteins to detect similarities. Such similarities are clues to the evolutionary origin of a protein. For instance a viral protein which causes cancer in the host has been found to be nearly identical to a normal growth factor. Hemoglobin and myoglobin belong to the same family and the serine proteases define another group with common evolutionary origins.

§ 2. The aa sequence can give clues to the function of the protein. A leader sequence may be present or a sequence which indicates that it is to be glycosylated.

§ 3. The sequence allows the synthesis of a very specific probe for locating the gene which synthesized the protein in whole genomic DNA.

1. Edman degradation

§ In this procedure a reagent is added which reacts specifically with the N terminal amino group on the polypeptide. The reacted polypeptide is then treated so as to release only the terminal amino acid residue which is coupled to the reagent. The terminal amino acid can be separated chromatographically from the remainder of the polypeptide.

Repeated rounds of this can sequence a polypeptide 50 aa long.

Longer polypeptides must be broken into pieces by specific enzymes. The “overlap” allows the pieces to be put in the correct order. For example:

tryptic peptides chymotryptic peptide

ala-ala-trp-gly-lys val-lys-ala -ala-trp

thr-asn-val-lys

[thr-asn-(val-lys]-[ala-ala-trp)-gly-lys]

2. DNA sequencing

n If the nucleotide base sequence of the gene which makes a protein can be determined, the aa sequence of the protein can be deduced from a knowledge of the genetic code.