Protein Biochem IV

MBG2007 Molecular and Cellular Biochemistry I

Lecture Xc - Lecture X11

Fall Semester 2025-2026
Instructor: Prof. Dr. Sezai Türkel

Methods in Protein Purification and Characterization - II

Overview

• This lecture focuses on methods used in protein purification and characterization, including assessment of protein quantity and purity.

References

1. Lehninger Biochemistry

2. Biochemistry, L Streyer

3. Biochemistry, J. Stenesh

Quantification and Purity Analysis of Proteins

Determination of Protein Amount

• Essential processes after protein purification include:

  • Quality control

  • Quantification

Spectrophotometric Measurement

• Principle: Measures light absorbance at specific wavelengths to determine protein concentration.

• Instrument Used: Spectrophotometer.

• Key Amino Acids: Tyrosine and Phenylalanine, absorbing UV light at ~280nm.

• Peptide Bonds: Absorb UV light around ~187nm.

• Common Methods for Protein Quantification:

  • Bradford assay (dye-binding method)

  • Lowry assay.

• Standard Protein: Bovine Serum Albumin (BSA) used to create standard curves for quantification.

Calculation of Protein Concentration

• Concentration can be calculated as follows:
  $\text{Concentration (mg/ml)} = \left(\frac{A<em>{595\ sample}}{A</em>{595\ std}} \times \text{Std conc}\ (mg/mL)\right)$

Bradford Method

Principle

• Based on changes in absorbance of Coomassie Brilliant Blue G-250 dye when it binds to proteins.

• Amino Acids Involved: Basic amino acids (Arginine, Lysine, Histidine) interact with the dye.

• Absorbance Shift: From 465 nm (free dye) to 595 nm (bound to protein).

Procedure

1. Mix protein sample with Coomassie dye in a small glass tube.

2. Wait for 5 minutes.

3. Measure absorbance at 595 nm using a spectrophotometer.

4. Compare the measurement to the BSA standard curve to determine protein concentration.

Example Calculation

• If the absorbance (A595) of an unknown sample is 0.5, use the standard curve to read the concentration as 1 mg/mL.

Lowry Folin-Ciocalteu Method for Protein Determination

Principle

• Based on the Biuret Reaction, enhanced sensitivity by additional reagents.

• Copper coats the nitrogen atoms in peptide bonds forming a cuprous complex.

• Folin-Ciocalteu Reagent: Interacts with cuprous ions producing a blue-green color detectable at wavelengths between 650 nm and 750 nm.

Sensitivity Range

• Protein Detection Range: 5–100 μg.

Ninhydrin Reaction (Amino Acid Analysis by TLC)

Process

• Limitations: Ninhydrin reacts with α + ɛ amino groups, ammonia nitrogen, leading to potential inaccuracies in quantification for proteins.

• Commonly used for amino acid identification in TLC or paper chromatography.

• Produces a purple product (Ruhemann's purple) at 540 nm, yellow product for proline at 440 nm.

Separation Techniques

• To separate amino acids effectively, techniques such as column chromatography (HPLC) may be used instead of SDS-PAGE electrophoresis.

Molecular Weights and Purity of Proteins

Monitoring Purification Process

• A purification table should be kept to document concentration and presence of proteins at every step.

• Common methods to check presence:

  • Enzyme Activity: Using relevant substrates.

  • ELISA Assay or Western Blot: Using specific antibodies.

SDS-PAGE for Purity and Molecular Weight Determination

• Purity Assessment: A single band on SDS-PAGE indicates 100% purity.

• Molecular Weight Determination: Through comparison with markers on SDS-PAGE gel. Different techniques can also be employed:

  • Density gradient centrifugation

  • Chromatography techniques.

• Amino Acid Composition: Can be used to approximate molecular weight (MW).

Factors Influencing Molecular Weight

• Post-translational modifications can alter MW, thus genetics and splicing events (e.g., alternative splicing resulting in isoforms) should be considered.

• Example: Alternative splicing of tropomyosin mRNA can yield 40 different isoforms.

Electrophoresis and Molecular Weight Estimation

Visualization Post-Electrophoresis

• Staining agents: Such as Coomassie blue visualize proteins post electrophoresis.

• Visualization Note: Smaller proteins migrate faster, appearing lower on the gel.

Estimation Techniques

• Electrophoretic mobility must be correlated with MW. Usage of standard proteins for calibration is essential:

  • Enable standards to approximate the MW of unknown proteins.

• Graphing: Plotting log MW vs. migration leads to linear relations aiding estimations.

End-Group Analysis

Overview

• Analytical determination involving terminal groups, originally developed by Fred Sanger for insulin.

Sanger Reaction

• Reagent: 1-Fluoro-2,4-dinitrobenzene (FDNB).

• Process: FDNB reacts with α-NH₂ groups forming DNP-amino acids. Followed by acid hydrolysis that cleaves peptide bonds.

• Separation and Identification: DNP-AA identified through chromatography due to distinct colors.

Dansyl Chloride Reaction

Introduction

• A method largely replacing the Sanger reaction due to lower toxicity and enhanced sensitivity.

• Detection: Dansyl derivatives exhibit fluorescence, enabling detection as low as 1 nanomole.

Edman Degradation

Principle

• Involves phenylisothiocyanate reacting with amino groups leading to phenylthiocarbamyl derivatives.

• Cleavage occurs at the N-terminal of proteins while preserving on-going peptide bonds.

Process Description

1. Initial treatment with phenylisothiocyanate.

2. Cleavage of the N-terminal amino acid occurs with anhydrous acid creates cyclic thiazolinone derivatives which can be separated for sequencing.

Automation and Sequencing

• Edman degradation is compatible with automation allowing for sequence determination of up to 40 amino acids in a peptide.

Cleaving Proteins into Peptides

Techniques

• Proteases: Enzymes that break peptide bonds adjacent to specific residues: e.g., trypsin acting on Lysine and Arginine.

• Reagents: Chemical methods may also cleave at defined sites.

Purpose of Peptide Cleavage

• Allows easier sequencing and analysis of large proteins by generating shorter, manageable peptides known as tryptic peptides.

Chemical Synthesis of Peptides

Merrifield Solid-Phase Method

• Developed by Robert Bruce Merrifield allows synthesis of long peptides attached to an insoluble support.

• Discoveries included automated peptide synthesizers that streamline peptide formation through sequential reactions.

Main Steps of Merrifield Synthesis

1. Carboxyl-end attachment of amino acids to a solid surface.

2. Activation of N-terminal

3. Peptide bond formation followed by washing of remaining free amino acids.

4. Deprotection of the N-terminal for further reactions.

Conclusion

• Protein purification and characterization techniques are crucial for molecular biology and biochemistry, impacting research and clinical applications. Understanding these methods enables effectiveness in various biochemical analyses and advancements in biotechnology.