Chapter 3 (part 2) Protein purification and Analysis

Why purify proteins?

• Pure proteins are required to study enzyme function • Pure proteins are required for structural analysis (x-ray crystallography, NMR spectroscopy) • Pure proteins are required to obtain amino acid sequence

Steps in protein purification • Develop assay • Choose source of protein • Prepare tissue extract – cell disruption – subcellular fractionation • Protein fractionation (several steps) • Determination of purity

Differential Centrifugation transfer supernatant transfer supernatant transfer supernatant

1000 g 10,000 g 100,000 g

tissue homogenate Pellet unbroken cells nuclei chloroplast Pellet mitochondria Pellet microsomal Fraction (ER, golgi, lysosomes, peroxisomes) Super.

Cytosol, Soluble enzymes

Chromatography

Gel Permeation Chromatography

Ion-exchange Chromatography + + + + + + + + + + + + + - - - - + + + + + + + + + + + + + + low salt buffer + + + + + - - + + + + + + + + + + + + + + + + + + + + + high salt buffer Cl + Cl + + + + + + + + + + + Cl Cl + Cl + + + + + + + + + + - - - - - - -

Affinity Chromatography Add excess ligand

SDS poly acrylamide electrophoresis (PAGE) SDS = H 3 C-(CH 2 ) 10 -CH 2 -OSO 3 SDS – denatures protein coats w/ negative charge - Used to determine protein MW And purity of protein prep

pH 9 Isoelectric Focusing pH 3 + +

large small 2-D Electrophoresis Decreasing pH SDS-PAGE + Decreasing pH

Amino Acid Analysis 1) Acid hydrolyze protein 2) Treat with phenylisothiocyanate (PICT) N C S + H 3 N H C O C O R N S C HN C C R H O 3) Separate derivatized AA’s by HPLC

1) Protein Sequencing (Edman Degradation) H O N C S H 3 N C C NH H C O C R R X 2) N S C HN H C O C H C O C X NH 3) R R Trifluoroacetic acid N S C C HN C R H + 2HN H C R O C X O Can sequence in 30 to 60 AA’s from N-terminus

Generate Proteolytic Fragments Endopeptidases •Typsin •Chymotrypsin Chemical Cleavages cleaves at COOH end of Lys and Arg cleaves at COOH end of Phe, Tyr, Trp •Cyanogen Bromide cleaves at COOH end of Met Generate overlapping fragments Sequence individual fragments and piece together sequence

Peptide mapping exercise Met-Ala-Arg- Gly-Glu-Tyr-Met-Cys-Lys-Phe-Ala-Glu-Gln-Asp Trypsin Met-Ala-Arg Phe-Ala-Glu-Gln-Asp Gly-Glu-Tyr-Met-Cys-Lys Chymotrysin Met-Ala-Arg- Gly-Glu-Tyr Met-Cys-Lys –Phe Ala-Glu-Gln-Asp CNBr Met Ala-Arg-Gly-Glu-Tyr-Met Cys-Lys-Phe-Ala-Glu-Gln-Asp

Proteomic Analysis

M atrix A ssisted L aser D esorption I onization T ime o f F light (MALDI-TOF)