Question

procedure to determine the purity of protein by spectroscopy

Answer 1

Estimation of protein concentration in a given protein preparation is one of the most
commonly performed tasks in a biochemistry lab. There are several ways of
estimating the protein concentration such as amino acid analysis following acid
hydrolysis of the protein; analyzing the changes in the spectral properties of certain
dyes in the presence of proteins; and spectrophotometric estimation of the proteins in
near or far UV region. Although dye-binding assays and amino acid analysis
following acid hydrolysis of the protein can be used for estimating the protein
concentration for both pure as well as an unknown mixture of proteins; UV
spectroscopic quantitation holds good for the pure proteins. If a protein is pure, UV
spectroscopic quantitation is the method of choice because it is easy and less time-
consuming to perform; furthermore, the protein sample can be recovered back.
Absorption of ultraviolet radiation is a general method used for estimating a large
number of bioanalytes. The region of the electromagnetic radiation ranging from ~10
– 400 nm is identified as the ultraviolet region. For the sake of convenience in
referring to the different energies of UV region, it can be divided into three regions:
• Near UV region (UV region nearest to the visible region; λ ~ 250 – 400 nm)
• Far UV region (UV region farther to the visible region; λ ~ 190 – 250 nm)
• Vacuum UV region (λ < 190 nm)
This division is not strict and you may find slightly different wavelength ranges for
these regions. We shall, in this course, stick to the above-mentioned definitions.
Absorption of UV light is associated with the electronic transitions in the molecules
from lower to higher energy state