PROTEIN PURIFICATION AND ANALYSIS

Assays Need measures for the object (enzyme activity, chromophore, etc.) and for total protein concentration:

Use spectrophotometry for: 1.Measuring substrate or product concentration if one is “colored” (absorbs UV or visible light). 2. Measuring substrate or product concentration if one reacts with something to form a colored compound. 3.Measuring the concentration of total protein (by UV absorbance: A 280 ~ 1 mg/ml) or of a specific protein (like hemoglobin) that contains a colored “chromophore.” 4.Measuring the concentration of protein by reacting it with a reagent to form a colored compound. (E.g., cys, tyr, trp reduce Cu 2+ to Cu +, which forms an intense blue with BCA; Coomassie reagent turns blue in hydrophobic environment.)

Measuring purification Specific activity (SA) = total enzyme activity/total protein or activity concentration/protein concentration 1 IU (international unit) = 1 µmol substrate used (product formed)/min-mg protein “Purification” at step x = SA of step x / SA of crude extract One measure of “purity” is constant SA. Note: if the protein of interest is 0.2% of the total, then you require 500-fold purification for purity; if each step gives 3-fold purification, then you need 5-6 steps; if each step causes some loss of the protein of interest (e.g. 50%), you need to start with times more enzyme than you will recover.

Purification techniques: size, charge, specific binding Size: exclusion chromatography

Two-dimensional gel electrophoresis: IEF followed by SDS-PAGE

Affinity chromatography: separate by biological specificity 1. Attach “ligand” to insoluble column material 2. Combine protein mixture with column: enzyme binds to ligand 3. Wash column to remove unwanted material 4. Elute enzyme with substrate or change in pH (to reduce binding)

Summary Enzyme purification involves: breaking cells and stabilizing pH, proteolysis, measuring enzyme activity and protein concentration, a series of separation techniques Spectrophotometry is useful for measuring protein concentration and, often, enzyme activity Exclusion chromatography, electrophoresis, isoelectric focusing, and affinity chromatograph are useful methods for purifying proteins