Protein Extinction Coefficient: Key Parameter in Bioanalysis

The extinction coefficient of protein, also known as molar extinction coefficient, is a parameter that measures the absorbance capability of protein at a specific wavelength. It is crucial for bioanalysis, especially in the determination of protein concentration, assessment of protein purity, and the study of protein interactions with other molecules.

 

Definition

The protein extinction coefficient (also known as the molar extinction coefficient) represents the absorbance of a 1 cm thick solution of protein at a concentration of 1 M at a specific wavelength. Its commonly used units are M^-1cm^-1 or L mol^-1cm^-1.

 

Importance

1. Protein Concentration Determination

By measuring the absorbance of a solution and using a known extinction coefficient, the concentration of protein can be accurately calculated.

 

2. Protein Purity Assessment

The extinction coefficient can help assess potential impurities or contaminants in protein samples.

 

3. Protein-Ligand Binding Studies

In the study of protein interactions with small molecules, drugs, or other proteins, the extinction coefficient can be used to calculate binding constants and thermodynamic parameters.

 

How to Determine the Extinction Coefficient

1. Theoretical Calculation

Based on the known protein sequence, its extinction coefficient at a wavelength of 280 nm can be predicted. This is mainly based on the content of tryptophan, tyrosine, and cysteine in the protein, as these amino acids have strong absorbance at 280 nm.

 

2. Experimental Determination

According to Lambert-Beer's law, when a monochromatic light passes through the flow pool, if the flow phase does not absorb the light, the absorbance A is directly proportional to the concentration C of the absorbing component and the light path length L of the flow pool. Therefore, the extinction coefficient of the protein can be calculated using the formula ε=A/cl, provided the absolute content of the protein and the extinction value of the test substance at a wavelength of UV 280 nm at a light path length of 1 cm is measured.

 

Precautions

1. Protein Status

The structure, conformation, or oxidation state of the protein may affect its extinction coefficient.

 

2. Impurities

Other substances in the sample, such as salts, solvents, or other impurities, may affect the absorbance measurement.