Separation and Characterization

Techniques for Proteins and Amino Acids

I. Protein IsolationSelection of Starting Material

• Sources– animal or plant tissues– microorganisms

• Criteria for choosing a sample– ease of obtaining sufficient quantity of

tissue– amount of biomolecule in the tissue– any properties peculiar to the

biomolecule of choice

Methods of Solubilization

Separation Techniques

Separation will be based on the characteristics of biomolecules.

• Solubility• Molecular size, weight, density• Affinity• Charge

Based on Solubility• Change in pH

Isoelectric precipitationA procedure in which the pH of the protein mixture is adjusted to the pI of the protein to be isolated to selectively minimize its solubility.

Isoelectric precipitation

• Change in ionic strengthSalting inSolubility of a protein at low ionic strength generally increases with the salt concentration.

Salting out Decrease in solubility of proteins and other substances in aqueous solution at high ionic strength. It is a result of the competition between added salt ions and other dissolved solutes for molecular solvation.

Based on Molecular Size

CentrifugationProcess of subjecting a suspension of

sample at greatly increased gravitational field (centrifugal force) by rapidly rotating a receptacle containing the sample which will lead to sedimentation of particles.

Application:Differential Centrifugation

For separation of crude mixtures of cellular components

Dialysis

• It is the movement of molecules by diffusion from high concentration to low concentration.

• A process that separates molecules by the use of a semi-permeable membrane.

Ultrafiltration

• When macromolecular solution is forced under pressure thru a semi-permeable bag/disc.

Gel Filtration Chromatography

• Column is packed with porous beads

• Small molecules enter the beads and are retarded, while, large molecules cannot enter and so they migrate faster

Based on Affinity

Chromatography• Separation of molecules in a

mixture depends on the affinity to either mobile or stationary phase.

• Types of Chromatography based on the polarity of each phase:

• Normal phase chromatography• Reverse phase chromatography

Affinity Chromatography

A procedure based on the ability of proteins to interact with specific molecules.

Based on Charge

• Electrophoresis– It is the separation of charged

particles in an electric field thru a support medium.

Isoelectric focusing

• Involves electrophoresis of protein mixtures thru stable pH gradient medium.

• Protein will migrate to the region where pH = IpH.

Gel Electrophoresis

Types of Gel:

1. Agarose

2. Polyacrylamide

SDS-PAGE

• SDS: mask the intrinsic charge of protein due to large negative charge it imparts on it.

• Separates protein in the order of their MWs.

Ion Exchanger Chromatography

• Similar to affinity chromatography

• Interaction is based on net charge

• Column is packed with resin that have ligand (either positive or negative in charge)

Cation Exchanger

Anion Exchanger

Determination of 10 Structure of ProteinA. Qualitative and Quantitative Analysis of Amino

Acids1. Hydrolyze peptide with 6N HCl at 110 C for 24 hours2. Separate mixture by an amino acid analyzer

B. Determination of Amino Acid Sequence1.Methods for identification of N-

terminal amino acid residuea. Sanger’s Reagent (DNFB)

b. Dabsyl chloride and Dansyl chloride

c. Edman Degradation

• Sequentially removes one residue at a time from the amino end.

• Phenyl isothiocyanate reacts with amino group to form a phenylthiocarbamoyl derivative.

• Mild acid conditions create cyclic derivative

• Cyclic derivative is separated by chromatography to identify amino acid

d. Aminopeptidase

Gly – Arg – Phe – Ile – Lys – Met – Leu

2. Methods of Identification of C-terminal amino acid residuea. Carboxypeptidase

Gly – Arg – Phe – Ile – Lys – Met – Leu

b. Hydrazinolysis

3. Cleavage of Protein into Peptides a. Chemical Method

Cyanogen bromide – cleaves at carboxyl side of Met

b. Enzymatic MethodTrypsin - cleaves on carboxyl side of Arg and Lys, but not when Pro is presentChymotrypsin - cleaves on carboxyl side of aromatic amino acid, but not when Pro is present