Determination of total protein content based on protein light absorption properties

Summary

Determination of total protein content based on protein light absorption properties is based on the light absorption properties of different chemical bonds of proteins. At present, there is one main method to determine the total protein content based on the light absorption properties of proteins: for example, the ultraviolet spectral absorption method.

Principle

The basic principle of UV spectroscopic absorption is Proteins have two absorption peaks in the ultraviolet region. One absorption peak is at 280nm, which is caused by the conjugated double bond of the benzene ring in the tryptophan and tyrosine molecules of the aromatic amino acids in proteins, of which tryptophan has the strongest light absorption, followed by tyrosine. The aromatic amino acid content of most proteins does not vary much, so the absorbance of the solution at 280nm can be used to derive the protein content. The UV absorption of relatively purified protein solutions without nucleic acid contamination (A ₂₈₀／A ₂₆₀) is about 1.8, which is too low to indicate the presence of high levels of nucleic acid impurities. Proteins in the ultraviolet region of the other absorption peak is caused by peptide bonds, in the 240nm below the time density increases sharply, 215nm at the absorption rate of several times the 280nm. The difference between the absorbance at 215nm and 225nm can be used to determine the protein content of very low protein solutions.

Operation method

Determination of total protein content by ultraviolet spectral absorption method

Materials and Instruments

Equipment:
UV spectrophotometer, quartz colorimetric cups
Reagents:
①
Standard solution of known protein content

Move

The basic process of determining the total protein content by UV spectral absorption can be divided into the following steps:

A. Take a volume of appropriately diluted protein solution, place it in a quartz colorimetric cup, and read the absorbance at the appropriate wavelength on a UV spectrophotometer. The absorbance reading should be in the range of 0.1 to 0.8, below or above which large errors will occur.

B. There are two methods of calculating the protein content of a solution from absorbance readings. One is to make a standard curve based on the UV-absorbed optical density of a standard solution with known protein content, and then compare the sample to the standard curve to calculate its protein content. Another method is to estimate the protein content based on the A280/A260 ratio of the solution using the following empirical formula:

Protein concentration ( mg/ml) = 1.45xA280-0.74xA260

Caveat

1. When applying the ultraviolet spectral absorption method to determine the protein content, it must be noted that the liquid used to zero the instrument should be the same as the sample to be measured, and the same solvent should be used for standard proteins, in order to avoid the ultraviolet absorption characteristics of the solvent to interfere with the sample determination. Table 10-2 summarizes some of the characteristic data of several protein content determination methods, which can be selected according to the specific situation.

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