Chapter 3: Amino Acids, Peptides, and Proteins

3.3-3.4

ion exchange

• based on charge of protein

• proteins carry different net charges depending on amino acid composition/pH of buffer

• column has charged resin beads

types of ion exchange

cation exchange, anion exchange

cation exchange (ion exchange)

• resin is negatively charged and binds positively charged proteins

• proteins with large negative net charge will be first to elute ( - > 0 > +)

anion exchange (ion exchange)

• resin is positively charged and binds negatively charged proteins

• proteins with large positive net charge will be first to elute (+ > 0 > -)

how to elute resin-bound proteins

• increase salt concentration (add NaCl)

  • salt ions compete/win in being bounded to resin; postive proteins elute

• pH change

  • alter protein’s pI

  • when protein no longer carries charge opposite to resin, it releases

• pH/salt gradient

  • dialysis follows (NH4)2SO2 fractionation to remove salt prior to chromatography

gel filtration

• based on size of protein

• column has porous beads

• large beads are too big to enter beads → elute first

• smaller beads take longer because they go through beads → elute last

affinity chromatography

• based on specific binding affinity between protein + ligand

  • stationary phase has ligand (substrate/cofactor) that specifically binds to target protein 

  • non-binding proteins are washed away

  • bound protein is eluted by

    • adding competing ligand, changing pH, or altering ionic strength

specific activity

purity of enzyme (enzyme activity/protein concentration) (mg/mL)

assay

reaction of protein with chemical reagent that produces color proportional to protein concentration

• standard curve is made using known protein (ex. BSA)

• absorbance of unknown samples is compared to curve to determine concentration

• color intensity (absorbance) is directly proportional to protein concentration

assay example

Bradford assay (595 nm)

• Coomassie Brilliant Blue dye binds to basic/aromatic residues; red → blue

• very fast, sensitive, and widely used; compatible with many buffers

absorbance spectroscopy

proteins naturally absorb UV light due to aromatic amino acids (Y, F, W) and peptide bonds

absorbance spectroscopy at 205 nm

• absorbance at 205 nm: 

  • peptide bonds absorb strongly here

  • useful if protein has few aromatic residues, but method is less specific because many buffers absorb at 205 nm

absorbance at 280 nm

• absorbance at 280 nm:

  • aromatic residues absorb strongly at 280 nm

  • directly measure protein concentration (A=εcl)

gel electrophoresis

movement of charged particles in electric field

• bands correspond to different sizes/charges

gel electrophoresis steps

1) proteins/nucleic acids are placed in gel matrix

2) when electric current is applied:

• negatively charged molecules move towards anode (positive)

• positively charged molecules move towards cathode (negative)

3) smaller molecules move faster

4) after separation, bands are stained with Coomassie dye for visualization

SDS-PAGE

molecular size/mass only

• estimates protein size/purity

SDS-PAGE steps

1) SDS denatures proteins and coats them with negative charge, masking their native charge differences

2) DTT can be added to break disulfide bonds, making proteins same

3) when electric field is applied:

• all proteins migrate towards positive cathode

• smaller proteins move faster through gel pores

4) gel is stained to visualize bands

5) each band corresponds to protein of particular molecular weight

isoelectric focusing

pI - pH at which protein has no net charge

isoelectric focusing steps

1) gel is prepared with stable pH gradient (low → high pH)

2) when voltage is applied:

• proteins migrate according to net charge

  • acidic/negative proteins move toward anode (+)

  • basic/positive proteins move toward cathode (-)

• proteins stop moving when pI=pH

3) proteins are separated by their isoelectric points

2D gel electrophoresis

pI + size

• combines IEF and SDS-PAGE

2D gel electrophoresis steps

1) isoelectric focusing

• proteins are separated in tube/strip gel based on pI

2) SDS-PAGE

• proteins are separated by molecular weight

3) protein appears as spot on 2D gel

• horizontal position: pI

• vertical position: molecular weight

sequencing small proteins (<50)

1) reduce/denature disulfide bonds and then alkylate Cys residues

2) determine amino acid composition via hydrolysis with 6 M HCl then analysis via HPLC

3) determine identity of N-terminal residue either via

• treat with Sanger’s reagent (FDNB), then do acid work-up (rest of peptide destroyed)

• do first step of Edman degradation using phenylisothiocyanate; this liberates one amino acid at a time (rest of peptide intact)

4) sequence by Edman degradation

sequencing large proteins (>50)

1) complete steps 1-4 for small proteins

2) digest protein into smaller residues suitable for Edman degradation via proteases

• Trypsin cleaves on C side of R/K

• Chymotrypsin cleaves on C side of F/W/Y

• cyanogen bromide (CNBr) cleaves on C side of Met

3) sequence by Edman degradation on each fragment; deduce amino acid sequence from overlaps

trypsin cleaves on C side of __

arginine (R), lysine (K)

chymotrypsin cleaves on C side __

phenylalanine (F), tryptophan (W), tyrosine (Y)

cyanogen bromide cleaves on C side of __

methionine (M)

Why must long polypeptides be broken into sets of peptide fragments for sequencing?

• sequencing methods can’t handle very long chains at once

• overlapping fragments are needed to construct full sequence

• smaller fragments are easier to chemically handle/analyze

• improves accuracy/resolution in both chemical/mass spec techniques

how to determine sequence of polypeptide, given info such as N-terminal PTH adduct structure and proteolytic digest results

1) start with any direct N-terminal Edman data

2) list cleavage specificities used in digests

• trypsin: C side of K/R

• chymotrypsin: C side of P/W/Y

• CNBr: C side of M

• DTT: reduces difsulfide bonds

• Edman’s reagent: determines amino-terminal amino acid in a polypeptide

• 6 M HCl: determination of amino acid composition of polypeptide 

3) obtain peptide fragments produced by each digest; fragments can be identified using direct sequencing, mass spec, or combination

4) make inventory of fragments from each digest

5) build overlaps

6) assemble full sequence by joining overlapping fragments into consistent linear order

7) check consistency

DTT function

reduces disulfide bonds

6 M HCl

determines amino acid composition of polypeptide