Q: Checking DNA purity using spec...

[Dr S. Robertson]

Otter (otterpop at pilot.msu.edu) wrote:
: I have been using the standard protocol to determine isolated DNA purity by
: measuring the light absorbance at 260 and 280 nm.  I know that pure DNA
: absorbs at a wavelength of 260 and protein at a wavelength of 280.  My
: questions is what exactly or specifically is absorbing the light at those
: wavelengths (i.e. does one base absorb more light than another at 260, are
: there specific amino acids that are absorbing at 280 etc...)?

: Thanks for your time and help,

: Otter

--

Proteins contain three amino acids which contribute to their absorbance at 
280nm. These are Tryptophan (Trp), Tyrosine (Tyr) and Cystine (Cys), although 
most of the absorbance is due to Trp which actually has an absorbance max of 
280nm or very near. Tyr absorbs considerably less and Cys less than that. 
Neither actually absorb at 280nm, one is a bit above the other a bit below 
(though for the life of me I can't remember which way round). This gives the 
UV spectra of proteins their characteristic shape with a shoulder either side 
of the main peak. In fact the extinction coefficient of a protein can be 
calculated with reasonable accuracy from just the number of these three 
residues in the sequence. ( Gill and von Hippel (1989) Anal. Biochem., 182, 
319-?).

There is little doubt that the bases of DNA each absorb slightly differently 
although in general it is assumed that there is no difference and a mean is 
taken which in most cases is accurate enough.

Hope this helps

Stephen Robertson
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| Stephen Robertson, University of Liverpool, School of Biological Science, |
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