Lec 3 Determining Protein Concentration

Purification Steps:
- Chicken breast muscle is the source of lactate dehydrogenase (LDH).
- Homogenization: Process to break down the tissue.
- Centrifugation: Separates cellular debris from soluble proteins.
- Ammonium Sulfate Precipitation: Increases protein concentration by precipitating proteins out of solution.
- Centrifugation: Used again to separate precipitated proteins from the solution.
- Desalting Chromatography (G-25): Removes small molecules like salts from the protein solution.
- Pseudo-affinity Chromatography (Cibacron Blue Agarose): A method to purify proteins based on their binding affinity.
Downstream Assays:
- Enzyme Assay: Measures enzyme activity.
- Electrophoresis: Used to check protein purity and analyze protein profiles.
- Enzyme Kinetics: Study of the rates of enzyme-catalyzed reactions.
Important Tasks:
- Calculate enzyme specific activity.
- Save samples at different purification steps.

Two commonly used methods in class:
1. Bradford Assay
2. Warburg-Christian Method

A classic method for protein quantification widely used in laboratories.
Mechanism:
- Based on the absorption of Coomassie (Brilliant) Blue dye by proteins.
- The dye changes from yellow at basic pH to blue at acidic pH (maximum absorbance at 595 nm).
Binding Characteristics:
- Dye binds through hydrophobic interactions and ionic bonds to ammonium group-containing side chains (Lys, Arg, His).
Sensitivity:
- Detection range: approximately 1 – 20 μg of protein.

Coomassie G-250 dye exists in three forms:
- Anionic (blue): predominant form bound to protein at pH levels below 3.
- Neutral (green): present in solution but weakly absorbs light.
- Cationic (red): another form present at higher pH levels.
Maximum absorbance characteristics:
- Anionic form has absorption maximum at 595 nm.
- Binding causes a shift from 465 nm (free dye) to 595 nm (bound form).

Interfering Factors:
- Basic conditions and detergents such as SDS can hinder binding efficiency, leading to underestimation of protein concentrations.
- The assay reaction is time-dependent, requiring simultaneous analysis of standards and unknown samples for consistent results.

A standard curve is essential to correlate absorbance values (A595) with protein concentrations.
The curve should exhibit linear relationships.

Overview: An older method based on the absorbance of Trp and Tyr residues in proteins.
Mechanism:
- Proteins absorb UV light at 280 nm and, to a lesser degree, 260 nm due to presence of aromatic amino acids.
Advantages:
- Fast and easy to perform.
- Ratio of A280/A260 can provide estimates for nucleic acid or protein concentrations in respective samples.
Disadvantages:
- Depends on the proportion of Trp and Tyr in proteins.
- Less sensitive than the Bradford assay, measurement range of 50-2000 μg/ml.

Function: Measures electromagnetic energy transmitted through a solution.
Key Formula: E = h\nu = \frac{hc}{\lambda}
- Where:
- h: Planck’s constant
- \nu: frequency
- c: speed of light
- \lambda: wavelength
Biochemical Relevance:
- Assessed wavelength range between 200-800 nm is crucial for observing molecular transitions.

Describes the relationship between absorbance (A), molar extinction coefficient (ɛ), path length (l), and concentration (c):
A = \varepsilon \cdot l \cdot c
Explanation:
- Caused by the amount of light absorbed proportional to the concentration of the absorbing species and the pathlength of the sample.

Conjugated Biomolecules: Detectable via absorption spectra.
Metalloproteins: Exhibits unique spectral properties (e.g., cytochrome c).
Nucleic Acids: Can also be quantified using spectrophotometry, primarily through absorbance at 260 nm.

Absorbance Ratios (A280/A260) and Correction Factors to estimate nucleic acid percentages.

Shows absorption maxima and extinction coefficients for various amino acids in neutral solutions.