Question

If we observe in a protein solution that DNA is causing a mixture 260/280 ratio ~0.9, and the 260/280 ratio for pure protein is about 0.6,

What happens to this 260/280 ratio as we remove the DNA from the protein solution?

Answer 1

As per some of the researchers, if protein has ratio higher than 0.7, it indicates nucleic acid contamination as discussed above (protein solution is contaminated by DNA and has brought ratio upto 0.9). Most of the researchers are suggesting to remove contaminants by using enzymes. It will help in lowering the ratio upto 0.6 and provide accurate results.

Source: https://www.researchgate.net/post/Why_do_I_get_A260_280_absorbance_value_105_of_protein_solution

Nucleic acids have absorbance maxima at 260 nm. Historically, the ratio of this absorbance maximum to the absorbance at 280 nm has been used as a measure of purity in both DNA and RNA extractions. A 260/280 ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA (Source: http://www.mgp.cz/files/nanodrop/manualy/pomer_cistoty.pdf)

Proteins in solution absorb ultraviolet light with absorbance maxima at 280 and 200 nm. Amino acids with aromatic rings are the primary reason for the absorbance peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can alter the absorbance spectrum. (Source: https://www.ruf.rice.edu/~bioslabs/methods/protein/abs280.html).

So as explained above there is range for determination of pure DNA, RNA and proteins.  Aromatic amino acids are relatively nonpolar. To different degrees, all aromatic amino acids absorb ultraviolet light. Tyrosine and tryptophan absorb more than do phenylalanine; tryptophan is responsible for most of the absorbance of ultraviolet light 280 nm by proteins. Tyrosine is the only one of the aromatic amino acids with an ionizable side chain. Tyrosine is one of three hydroxyl containing amino acids. (http://www.biology.arizona.edu/biochemistry/problem_sets/aa/Aromatic.html)

Most probably ratio will be same if tryptophan, tyrosiene and cysteine are present in proteins as these have absorbance at 280nm, Here ratio will not change as value of 0.6 is given as purified valuefor proteins. But if other proteins are present then value can vary from 200nm and above. If above 3 proteins are not present then their value can be determined by Scopes method in which wavelength of 205nm is used. (http://www.microspectra.com/support/learn/protein-absorption).

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