UV Protein Determination.

Ron Kagan rkagan at ewald.mbi.ucla.edu
Fri Sep 23 19:27:49 EST 1994


In article <ckuan.780273269 at sfu.ca> Min Kuan, ckuan at kits.sfu.ca writes:
> 	I am doing some UV protein scanning to esterminate protein 
>concentration of purified alpha-chymotrpsin, and crude alpha-chymotrpsin 
>that has Bovine Serum Albumin (BSA) in a ratio of 1:1 from 
>280nm to 340nm.   
>
>	According to an assay method on this enzyme described in 
>Worthington Enzyme Manual (1972, p129), the calculation given is
>as followed:
>
>		Extinction coefficient: E(1%, 280) = 20.4
>
>		mg/ml = A(1cm,280nm) x 0.49
>	        ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^	I use this underlined
>formula to calculate the protein concentration for the purified protein,
>right? The problem is, how do I find out the extinction coefficient
>for the Crude Enzyme? I can't use the E for the purified enzyme (20.4)
>since the crude one has BSA in it. 
>
>	So, any ideas?    :)

You could consider that the absorbance that you are getting at 280 nm is
the *sum* of the absorbances for BSA and chymotrypsin.  You could then
look up the extinction coefficient for BSA in the CRC Biochemistry
handbook, or the Merck index, or some source like that.  If you know in
advance that your "crude" mixture is merely a 1:1 mixture of both
proteins (with no other abosrbing species) then you should be able to
easily write out an equation that allows you to calculate the
concentration of chymotrypsin in the mixture!

Regards,
Ron

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"My Kindness is not random, and nothing senseless was ever beautiful."

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Ron Kagan
rkagan at ewald.mbi.ucla.edu



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