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How Are Proteins Isolated and Purified from Cells?

• The thousands of proteins in cells can be separated and purified on the basis of size and electrical charge • Proteins tend to be least soluble at their isoelectric point • Increasing ionic strength at first increases the solubility of proteins (salting-in), then decreases it (salting-out)

How Is the Amino Acid Analysis of Proteins Performed?
• Acid hydrolysis liberates the amino acids of a protein • Note that some amino acids are partially or completely destroyed by acid hydrolysis • Chromatographic methods are used to separate the amino acids • The amino acid compositions of different proteins are different

How is the Primary Structure of a Protein Determined? • The sequence of amino acids in a protein is distinctive • Both chemical and enzymatic methodologies are used in protein sequencing

Frederick Sanger was the first - in 1953, he sequenced the two chains of insulin. • Sanger's results established that all of the molecules of a given protein have the same sequence. • Proteins can be sequenced in two ways: - real amino acid sequencing - sequencing the corresponding DNA in the gene

Determining the Sequence An Eight Step Strategy

• 1. If more than one polypeptide chain, separate. • 2. Cleave (reduce) disulfide bridges • 3. Determine composition of each chain • 4. Determine N- and C-terminal residues

Determining the Sequence An Eight Step Strategy

• 5. Cleave each chain into smaller fragments and determine the sequence of each chain • 6. Repeat step 5, using a different cleavage procedure to generate a different set of fragments.

Determining the Sequence An Eight Step Strategy • 7. Reconstruct the sequence of the protein from the sequences of overlapping fragments • 8. Determine the positions of the disulfide crosslinks

Step 1:
Separation of chains • Subunit interactions depend on weak forces • Separation is achieved with: - extreme pH - 8M urea - 6M guanidine HCl - high salt concentration (usually ammonium sulfate)

Step 2:
Cleavage of Disulfide bridges • Performic acid oxidation • Sulfhydryl reducing agents - mercaptoethanol - dithiothreitol or dithioerythritol - to prevent recombination, follow with an alkylating agent like iodoacetate

Step 3A:
Identify N- and C-terminal residues • N-terminal analysis: – Edman's reagent – phenylisothiocyanate – derivatives are phenylthiohydantoins – or PTH derivatives

Step 3B: :
Identify N- and C-terminal residues • C-terminal analysis – Enzymatic analysis (carboxypeptidase) – Carboxypeptidase A cleaves any residue except Pro, Arg, and Lys – Carboxypeptidase B (hog pancreas) only works on Arg and Lys

Steps 4 and 5:
Fragmentation of the chains • Enzymatic fragmentation – trypsin, chymotrypsin, clostripain, staphylococcal protease • Chemical fragmentation – cyanogen bromide

Enzymatic Fragmentation
• Trypsin - cleavage on the C-side of Lys, Arg • Chymotrypsin - C-side of Phe, Tyr, Trp • Clostripain - like trypsin, but attacks Arg more than Lys • Staphylococcal protease – C-side of Glu, Asp in phosphate buffer – specific for Glu in acetate or bicarbonate buffer

Chemical Fragmentation
Cyanogen bromide • CNBr acts only on methionine residues • CNBr is useful because proteins usually have only a few Met residues

Reconstructing the Sequence
Compare cleavage by trypsin and staphylococcal protease on a typical peptide: • Trypsin cleavage: A-E-F-S-G-I-T-P-K L-V-G-K • Staphylococcal protease: F-S-G-I-T-P-K L-V-G-K-A-E

Reconstructing the Sequence
L-V-G-K A-E-F-S-G-I-T-P-K F-S-G-I-T-P-K • Correct sequence: L-V-G-K-A-E-F-S-G-I-T-P-K L-V-G-K-A-E

Amino Acid Sequence Can Be Determined by Mass Spectrometry • Mass spectrometry separates particles on the basis of mass-to-charge ratio • Fragments of proteins can be generated in various ways • MS can also separate these fragments

What is the Nature of Amino Acid Sequences?
• Sequences and composition reflect the function of the protein • Membrane proteins have more hydrophobic residues, whereas fibrous proteins may have atypical sequences • Homologous proteins from different organisms have homologous sequences • e.g., cytochrome c is highly conserved