Electrophoresis Electrophoresis is the migration of ions in an

electric field. Electrophoresis of proteins is typically carried

out in agarose or polyacrylamide gels with characteristic pore size. The molecular separation is therefore based on both sieving effect and electrophoretic mobility.

Note: The term anode and cathode are often used in electrophoresis and are often confused. Remember that an anion is a negatively charged ion; anions move to the anode and vice versa.

Sample question

In what direction will the following proteins move in an electric field [toward the anode, toward the cathode or neither] Egg albumin (pHI = 4.6) at pH 5.0 -lactoglobulin (pHI = 5.2) at pH 5.0 and at pH

7.0?

Stabilising Proteins Isolation of a protein from its natural

environment, exposes it to many agents that can irreversibly (permanently) damage it. The following factors must be taken into consideration during the purification process:

pH: Buffer solutions of effective pH must be used at every stage. Failure to do so could result in denaturation which could lead to structural disruption and even chemical degradation.

Temperature: Protein purification is normally carried out at temperatures near 0oC.

Presence of degradative enzymes: These include nucleases and proteases that are released during cell disruption. These enzymes can damage proteins. Degradative enzymes are inhibited by adjusting the pH or temperature or by adding compounds that specifically block their action.

Adsorption to surfaces: Many proteins are denatured by contact with the air-water interface or with glass or plastic surfaces. Hence, protein solutions are handled so as to minimise foaming and are kept relatively concentrated.

Long-term storage: For long-term storage, slow oxidation and microbial contamination must be prevented. Protein solutions are stored under nitrogen or argon and frozen at -70 oC or at -196 oC (the temperature of liquid nitrogen).

Assaying for protein Purifying a substance in general requires some means for

quantitatively detecting it. Accordingly, assay must be devised that is specific for the target protein, highly sensitive and convenient to use (the protein of interest must be assayed for at every stage of purification).

Various techniques are used. Some assays are straight forward. e. g. assaying for enzymes that catalyse reactions with detectable products.

Other techniques include: coupled enzymatic reactions immunoassays. Examples of immunoassays are: radioimmunoassays

(RIA), enzyme-linked immunosorbent assays (ELISA).

Protein Structure Determination

The procedure can be divided into four stages: Primary structure determination. Secondary structure determination. Tertiary structure determination. Quaternary structure determination.

1O Structure

The primary structure of a protein is the sequence of its polypeptide chain or chains.

It also describes the amino acid composition of the protein.

It is the basic product of DNA transcription and translation.

It determines the nature of the subsequent folding of the polypeptide chain.

Summary

In summary, primary structure describes the types, number of individual amino acids in a protein and the order in which they occur.

Examples of primary structures For insulin:

Diversity of primary structure: Why are there so many polypeptide chains? There are 20 different amino acids and for a

protein of “n” residues, there are 20n possible sequences

Thus for relatively small protein molecule, consisting of a single polypeptide chain of 100 residues, there are 20100 ~ 1.27 x 10130 possible unique polypeptide chains of this length.

Actual size and composition of polypeptides

In general, minimum residue is 40. However, the vast majority of polypeptides contain between 100 and 1000 residues.

Polypeptide with many hundreds of residues may approach the limit of protein synthetic machinery.

The longer the peptide (and the longer its corresponding mRNA and its gene), the greater is the likelihood of introducing errors during transcription and translation.

Composition of some proteinsProtein aa residues No. of subunits Molecular mass (D)

Proteinase inhibitor III (melon) 30 1 3,409

Cytochrome c (human) 104 1 13,000

Ribonuclease H (E. Coli) 155 1 17,600

Interferon-(rabbit) 288 2 34,200

Haemoglobin (human) 574 4 64,500

RNA polymerase (bacteriophage T7) 883 1 98,000

Why determine primary structure? The knowledge of amino acid sequence is important for the

following reasons:

It is prerequisite for determining the three dimensional structure of a protein and for understanding its molecular mechanism of action.

Sequence comparisons among analogous proteins from different species give insight into protein function and reveal evolutionary relationships among the proteins and the organisms that produce them.

Many inherited diseases are caused by mutations leading to an amino acid change in a protein. Amino acid sequence analysis can assist in the development of diagnostic tests and effective therapies.

Determination of primary structure

It is the determination of the sequence or the order in which the amino acids occur in a protein.

The technique used in determining primary structure is called protein sequencing.

Protein sequencing

After satisfying oneself that the protein of interest is reasonably pure, the next step is to determine the amino acid sequence (primary structure).

The first protein to be sequenced was bovine hormone insulin by Frederick Sanger in 1953.

It took him over 10 years and require about 100g of protein. Procedures for primary structure analysis have since being refined and automated.

Techniques in protein sequencing

The technique is still similar to the procedure develop by Frederick Sanger more than 50 years ago:

Briefly, the protein is broken down into fragments small enough to be individually sequenced.

The primary structure of the intact protein is then reconstructed from the sequence of overlapping fragments.

Outline of procedure

Determine end groups Separate subunits if present Dislodge disulphide and other linkages Perform specific cleavage Sequence fragment Work out peptide sequence and then

positions of disulphide bonds

End group analysis:

N-terminal analysis and C-terminal analysis. This step is important because it gives idea of the

number of subunits that are in the protein to be sequenced.

For a multisubunit protein, the individual subunits must be separated and then sequenced.

Subunit separation is done by the cleavage of disulphide bonds (the main force holding subunits together).

N-terminal analysis This can be done by several methods:

Dansylation of N-terminal amino acid, followed by acid hydrolysis and identification of the dansylated residues by comparing with standards.

Edman degradation: It is a procedure that liberates aas one at a time from the N-terminus. N-terminal residues can be identified by performing the first step of Edman degradation.

Enzymatic hydrolysis: Aminopeptidases cleaves the N-terminal residues.

Dansylation 1-dimethylaminonaphthalene-5-sulfonylchloride (dansyl

chloride) reacts with primary amines (n-terminal aa) to yield dansylated polypeptides.

Acid hydrolysis liberates the modified n-terminal residue, which is separated chromatographically and identified by its intense yellow fluorescence.

The fluorescence is compared with standards to determine the n-terminal aa.

C-terminal analysis

There is no reliable chemical procedure for identifying the C-terminal residue of a polypeptide. However, this can be done by using

carboxypeptidases, enzymes that catalyse the hydrolytic excision of C-terminal residues.

Note: Aminopeptidases and carboxypeptidases are collectively referred to as exopeptidases.

Carboxypeptidases, like all enzymes, are highly specific for the chemical identities of their targets. For example, carboxypeptidase A, an intestinal digestive

enzyme, does not cleave C-terminal Arg or Lys residues or residues that are next to Pro.

Carboxypeptidase B, on the other hand, hydrolyses only Arg and Lys residues, but only if they are not preceded by Pro.

For this reason, the results of enzymatic end group analysis must be treated with caution. This is because if a carboxypeptidase cleaves the first residue slowly and the second quickly, its use may yield two amino acids, suggesting the presence of two different chains.

Cleavage of disulphide (S-S) bonds There are two types of S-S linkages:

a) interchain and b) intrachain.

Interchain S-S linkages are disulphide bonds between two Cys residues in two different polypeptide chains in a protein.

Intrachain S-S linkages, are disulphide bonds between two Cys residues in the same polypeptide chain.

There are two main reasons why S-S linkages must be cleaved:

To separate different polypeptide subunits. To make a polypeptide chain fully linear.

Disulphide bonds can be cleaved either oxidatively by performic acid or reduced by mercaptans (compounds containing –SH groups).

Performic acid oxidation

This was pioneered by Sanger. It involves treatment of the protein with performic acid.

It converts all Cys residues, whether linked by S-S bridges or not, to cysteic acid residues

Disadvantage of performic acid treatment It oxidises Met residues and partially destroys

the indole side chain of Trp.

Reductive cleavage of S-S links This is achieved by treatment with 2-

mercaptoethanol or any other mercaptan. Dithiothreitol; DTT (Cleland’s reagent) could also be used.

2-mercaptoethanol: HSCH2CH2OH DTT: HSCH2CH(OH)CH(OH)CH2SH

Reaction with Iodoacetic acid

In the reductive process, the –SH formed have the ability of forming the S-S bonds again.

They are therefore protected by alkylating the free sulfhydryl groups, usually by treatment with iodoacetic acid.

Cleavage of non-disulphide linkages Oligomers can contain subunits that are not linked

by disulphide bridges. Such subunits can be dissociated under acidic condition at low salt concentration and low temperature.

The dissociation could be achieved by treating the subunits with denaturising agents such as urea, guanidinium ion. One can also add detergents such as sodium duodecylsulphate (SDS). Urea: H2N-CO-NH2 Guanidinium ion: [C(NH2)3]+

Separation of subunits

The chemical separation of subunits in multisubunit proteins by either oxidative or reductive cleavage of disulphide bonds or cleavage of non-disulphide bonds, is followed by physical separation and purification using chromatography and/or electrophoresis.

Explain the following observation Electrophoretic separation of a protein A by

polyacrylamide gel electrophoresis, produces a band corresponding to 80 kD. When the band was cut, treated with urea and electrophoresis repeated, two bands were obtained at 30 kD and 50 kD.

Determination of amino acid composition In some cases, it is desirable to know the amino

acid composition of a polypeptide. The amino acid composition is the number of each

type of amino acid residue present in a polypeptide. The amino acid composition is determined by

complete hydrolysis of the polypeptide followed by the analysis of the liberated amino acids.

Peptide hydrolysis is accomplished by either chemical (acid or base) or enzymatic hydrolysis.

Acid, base or enzymatic hydrolysis None of these methods is alone fully satisfactory and are used to

complement one another.

Acid hydrolysis is achieved by dissolving the polypeptide in about 6 M HCl in a tube, which is then evacuated, sealed and heated to about 100-120 oC for about 10-100 hours. The disadvantage is that it degrades Ser, Thr, Tyr and Trp and converts Asn and Gln to Asp and Glu respectively.

Base hydrolysis is carried out in between 2-4 M NaOH at 100 oC for 4-8 hours. This process is even more problematic than acid hydrolysis. It causes decomposition of Cys, Ser, Thr, and Arg. Other aas undergo racemisation. It is therefore reserved for Trp which cannot be detected by acid hydrolysis.

Enzymatic hydrolysis is often incomplete and further complicated by the fact that the peptidases themselves are subject to proteolytic cleavage, thereby contributing to the total amino acid content.

Tryptophan is best determined spectrophotometrically in the unhydrolysed protein.

The components of the polypeptide hydrolysate are derivatised (either before or after chromatography) with an easily detectable tag.

Originally, the aas are separated by ion-exchange chromatography and quantified by post-column derivatisation to a coloured compound using ninhydrin.

Reverse-phase high performance liquid chromatography (HPLC) is now generally used. It is best achieved by pre-column derivatisation either by o-phthaladehyde or by phenylisothiocyanate. (Modern amino acid analyser can completely analyse a protein digest containing as little as 1 pmol of each aa in less than 1 hour).

Typical chromatogram

Polypeptide cleavage It involves cutting of the polypeptide chain into

smaller fragments. This procedure is generally referred to as “divide and conquer”.

It is achieved with the help of endopeptidases.

The peptide fragments generated by specific cleavage are isolated and sequenced.

Polypeptide cleavage cont’d

If an amino acid is not required to be lysed, that aa could be modified. That when a particular peptide bond needs to be maintained. E. g. The R group of Lys could be modified.