PROTEIN ISOLATION,

PURIFICATION, AND
CHARACTERIZATION
Nhyll Angelo S. Acuña
Protein/Enzyme Isolation

■ Isolating enzyme from its original environment (ie. plant tissues, microorganisms,
animal tissues).
■ Enzymes may be found:
– As secretion in the case of some microorganisms that release enzymes
extracellularly.
– As intracellular enzyme like on plants, animals, or some microorganisms, and
calls for cell lysis to liberate cytoplasm where enzyme resides.
– As membrane-bound enzyme, which are enzymes intact with the membrane
for certain purposes.
Protein/ Enzyme Purification

■ Step-by-step attempt to purify certain protein/enzyme of interest.

■ Involves certain techniques or combination of them for protein/enzyme purification
such as:
– Precipitation (Ammonium sulfate or Ethanol)
– Chromatography (Ion Exchange Chromatography, Size Exclusion
Chromatography, Hydrophobic Inhibition Chromatography, Affinity
Chromatography)
Protein/ Enzyme Purification

■ In absence of details other than functionality of an enzyme, it is handy to conduct an

activity guided assay to determine the enzyme of interest among the enzyme
fractions.
■ Certain provisions, however, must be followed in an activity-guided assay:
– Avoid enzyme denaturation agents like high temperature and extremes of pH.
– Ensure ample concentration of enzyme on the solution.
Protein/ Enzyme Purification

■ Concentration via freeze-drying or lyophilization may help you to concentrate your

enzyme while retaining its integrity (3D structure).
■ Dialysis may be also employed in the event of high salt concentration on your
mixture.
Protein Characterization

■ Determination of number of sub units and their sizes (Sodium Dodecyl

Polyacrylamide Gel Electrophoresis/ SDS-PAGE)
■ Determination of purity of protein (Native PAGE)
■ Determination of the present Amino Acid Identity and/or sequence (Mass
Spectrophotometry)
■ Determination of crystal structure of an enzyme (Atomic Absorption Spectroscopy)
Topics on Problem Sets (to be sent until
Friday)
■ Biochemistry applications
■ Protein Folding
■ Protein Isolation, Characterization, and Purification
■ Enzyme Kinetics
■ Inhibitors and Regulators
■ Henderson-Hasselbach Equation