March 10

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  • Copyright of Professor Omri

  • PowerPoint and lecture materials are exclusive to students enrolled in Biochemistry I (CHMI-2227 E) at Laurentian University, Winter term 2026.

  • Unauthorized or commercial use, including uploading to external sites, is prohibited.

Separation of Chains

  • Strong reliance on weak forces for subunit interactions.

  • Methods to achieve separation include:

    • Extreme pH

    • 8 M urea

    • 6 M guanidine hydrochloride

    • High salt concentration (commonly ammonium sulfate, (NH₄)₂SO₄)

Determining the Amino Acid Sequence of a Protein

Step 1: Separation of Chains

  • 8 M urea used for denaturation.

Step 2: Cleavage of Disulfide Bridges

  • Disulfide bonds between or within polypeptide chains must be cleaved.

  • Methods:

    1. Performic Acid Oxidation:

    • Converts all cysteine residues to cysteic acid residues.

    1. Sulfhydryl Reducing Agents:

    • Common agents: 2-mercaptoethanol, dithiothreitol, dithioerythritol.

Step 3: N-terminal and C-terminal Analysis

  • Preventing Recombination:

    • Cysteine residues are treated with alkylating agents, such as iodoacetate, to produce stable S-carboxymethyl derivatives.

  • N-terminal Analysis:

    1. Methods to identify the N-terminal residue:

    • Labeling all amino groups.

    • Hydrolyzing the peptide to amino acids.

    • Identifying the labeled amino acid.

    1. Pehr Edman Degradation:

    • Uses Edman’s reagent, phenylisothiocyanate (PITC).

    1. PITC Reaction:

    • Reacts with the N-terminal amino group of the peptide under alkaline conditions (pH = 9) to form a phenylthiocarbamoyl (PTC) polypeptide.

    1. Release of N-terminal residue:

    • Treatment with trifluoroacetic acid releases the N-terminal residue as a pyridine thiazolidinone derivative.

    • Further treating the thiazolidinone derivative with a weak acid gives a stable product called phenylthiohydantoin (PTH derivative), which is identified by chromatography.

    1. Repeat Analysis:

    • Remaining polypeptide (original length - 1) can be treated again with PITC for sequencing.

    1. Limitations:

    • Peptides longer than ~50 residues cannot be completely sequenced using Edman degradation.

Step 4: C-terminal Analysis

  • No reliable chemical methods for sequencing C-terminal amino acids.

  • Carboxypeptidases:

    • Carboxypeptidase A (from bovine pancreas) cleaves all amino acid residues except proline, arginine, and lysine.

    • Carboxypeptidase B (from hog pancreas) cleaves only C-terminal arginine and lysine residues.

Step 5: Fragmentation of Chains

  • Types of Fragmentation:

    • Enzymatic Fragmentation:

    • Enzymes used include trypsin, chymotrypsin, clostripain, staphylococcal protease, and endoproteinase Lys C.

    • Chemical Fragmentation:

    • Cyanogen bromide (CNBr).

  • Trypsin:

    • Cleaves on the C-side of lysine (K) and arginine (R).

  • Chymotrypsin:

    • Cleaves on the C-side of phenylalanine (F), tyrosine (Y), and tryptophan (W).

  • Clostripain:

    • Cleaves only at arginine (R).

  • Endoproteinase Lys C:

    • Cleaves only at lysine (K).

  • Staphylococcal Protease:

    • Cleavage occurs on the C-side of aspartate (E) and glutamate (D) in a phosphate buffer.

Step 6: Chemical Fragmentation with CNBr

  • Properties of CNBr:

    • Selectively cleaves the peptide bond on the C-side of methionine (M) residues.

    • Useful because proteins usually contain very few methionine residues.

    • The sulfur of methionine reacts with carbon of CNBr yielding a homoserine lactone at the C-terminal end of the fragment.

    • Resulting peptides are separated via chromatographic methods.

Reconstructing the Sequence

  • Utilize two or more fragmentation agents in separate experiments.

  • Sequence all generated peptides, typically through Edman degradation.

  • Compare and align overlapping peptide sequences to deduce the original polypeptide chain's sequence.

Example Comparison

  • Comparison of cleavage by trypsin and staphylococcal protease:

    • Original sequence: L-V-G-K-A-E-F-S-G-I-T-P-K

    • Trypsin cleavage (at R or K): L-V-G-K | A-E-F-S-G-I-T-P-K

    • Staphylococcal protease cleavage (at D or E): L-V-G-K-A-E | F-S-G-I-T-P-K

Structural Significance of Protein Sequence

  • Functional and structural properties of proteins depend immensely on the amino acid order in polypeptides.

  • Three-dimensional structure uniquely determined by amino acid sequence.

  • Genetic mutations affecting amino acid sequences can lead to diseases (Example: Delta F508 in cystic fibrosis).

  • Evolutionary conservation of amino acids points to their structural/function relevance.

Case Study: Sickle-Cell Anemia

  • Hemoglobin (Hb) consists of a tetramer formed by two types of polypeptides: HbA and HbB.

  • Sickle cell anemia results from a single mutation in the beta globin gene causing glutamic acid (Glu) to be replaced by valine (Val).

  • This mutation creates a hydrophobic patch, leading to abnormal aggregation of hemoglobin (HbS), forming long fibers that distort red blood cell shape.

  • Deformed red blood cells hinder blood flow and are fragile due to their shape, leading to anemia.

  • Interestingly, sickle cell anemia imparts a survival advantage against malaria, as the malaria parasite grows within erythrocytes.

  • Normal red blood cell vs. sickled red blood cell comparison highlights structural implications.