Xc XI Protein analysis 2

MBG2007 Molecular and Cellular Biochemistry I 2025-2026 Fall Semester

Lecture Overview

• Presented by Prof. Dr. Sezai Türkel

• Focuses on Methods in Protein Purification and Characterizations-II

References

1. Lehninger Biochemistry

2. Biochemistry by L. Streyer

3. Biochemistry by J. Stenesh

Quantification and Purity Analysis of Proteins

• Quality Control Post-Purification: After completing protein purification, quality control and quantification are essential steps.

Determination of Protein Amount

• A common method to measure the concentration of proteins involves using a spectrophotometer:

  • Measures the light absorption at specific wavelengths.

  • Key Absorbance Wavelengths:

  • 280 nm: Absorption peak for tyrosine and phenylalanine, key amino acids in proteins.

  • 187 nm: Peptide bonds absorb UV light but this is less common for quantitation.

Common Protein Quantification Methods

1. Bradford Assay (Dye binding assay)

   • Based on the principle that the absorbance of Coomassie Brilliant Blue G-250 changes when it binds to proteins.

   • Absorbance shifts from 465 nm (free dye) to 595 nm (bound dye) upon protein binding.

     • Highly suited due to its rapid execution and simplicity.

   • Standard curve preparation using Bovine Serum Albumin (BSA), which serves as a reference standard.

   • Calculating Concentration:

     • Use the equation:
       $Concentration ext{ (mg/ml)} = rac{A<em>{595 ext{ sample}}}{A</em>{595 ext{ std}}} imes ext{Std conc (mg/ml)}$

   • Example: If an unknown sample has an A595 value of 0.5, it can be quantified from a standard curve to give a concentration, e.g., 1 mg/mL.

2. Lowry Assay

   • Sensitivity: Enhanced detection with specific reagents.

   • It involves the biuret reaction where copper ions interact with nitrogen atoms in peptide bonds to form havable cuprous complexes.

   • Addition of Folin-Ciocalteu reagent reacts with these complexes creating a blue-green color detected between 650 nm and 750 nm.

   • Detection Range: 5-100 μg of protein.

Protein Analysis Techniques

Ninhydrin Reaction for Amino Acid Analysis

• Purpose: Visualizes location of amino acids on TLC plates as they are not inherently colored.

• Reaction with Ninhydrin:

  • Reacts with amino groups to form Ruhemann's purple dye that absorbs at 540 nm.

  • Proline is an exception, forming a yellow compound with absorbance at 440 nm.

• Limitations: Not preferred for protein quantification; mainly used for amino acid identification.

Determination of Molecular Weights and Purity of Proteins

• Monitoring Purification: It's essential to prepare a protein purification table. At every purification step, both protein presence and quantity are key.

• Techniques for verification include:

  • Using Bradford or Lowry assays for total protein quantification.

  • Enzymatic activity assays for specific proteins.

  • ELISA or western blotting for specific antibody recognition of target proteins.

  • SDS-PAGE:

  • Purpose: Assess protein purity and molecular weight; a single band indicates 100% purity.

  • Comparison with Marker Proteins: To establish molecular weights based on migration distances of reference proteins on the gel.

• Additional Techniques:

  • Density gradient centrifugation, chromatography, and amino acid composition analysis can also determine molecular weights.

Molecular Weight Approximation

• Genetic code and amino acid mass contribute to this calculation but note the potential for post-translational modifications affecting MW.

• Alternative Splicing: Results in varying molecular weights from the same gene, as seen with tropomyosin isoforms.

Electrophoresis and Visualization

• Use of SDS-PAGE:

  • Proteins denatured and separated on gel; smaller proteins migrate faster and toward lower gel sections.

  • Visualization with stains like Coomassie blue helps to identify protein bands corresponding to different polypeptides.

End-Group Analysis Techniques

• Used for analytic determinations focused on terminal groups of biopolymers.

Methods of End-Group Analysis

1. The Sanger Reaction:

   • Employs 1-fluoro-2,4-dinitrobenzene (FDNB) to react with N-terminal amino groups in alkaline solution.

   • Results in a DNP-amino acid, allowing for identification post acid-hydrolysis.

   • Identifiable via paper chromatography.

2. The Dansyl Chloride Reaction:

   • More sensitive than Sanger's method; dansyl derivatives are detectable at low concentrations.

   • Preferred due to the toxicity of FDNB.

3. The Edman Degradation:

   • Reacts with the terminal amino group using phenylisothiocyanate (PTC) under alkaline conditions.

   • Cleavage produces N-terminal derivatives, allowing sequential identification of amino acids.

   • The peptide chain is purified after each step of degradation to isolate targeted fragments.

Disulfide Bond Analysis

• Disulfide Cleavage: Essential for preparing large proteins for sequencing.

  • Can be achieved by oxidation or reduction methods.

  • Proteases such as trypsin hydrolyze specific peptide bonds adjacent to Lys or Arg residues, facilitating peptide generation.

Peptide Synthesis

• Merrifield Solid-Phase Synthesis:

  • Developed by Prof. Dr. Robert Bruce Merrifield in 1969 to synthesize ribonuclease.

  • Peptide chains synthesized attached to a solid support, automated in modern peptide synthesizers.

  • Each cycle involves the attachment and reaction of specific amino acids to form peptide bonds through carefully controlled steps.

  • Use of blocking groups (like Fmoc) and activation agents (like DCC) maximizes efficiency and accuracy.

• Final Process Overview: Ensures precise single amino acid additions through cycles leading to longer peptides, paving the way for complex sequencing analyses.