I am working on producing a protein called plastocyanin. The oxidized form of this protein can absorb light at 597 nm which distinguishes it from other proteins in the cell homogenate that absorb at 278 nm. After multiple steps of purification procedures, the absorbance ratio for my sample A278/A597 differs (pH denaturation 39.5, ion exchange 17.7, gel filtration 0.85). How can I empirically evaluate the purity of my protein based on this ratio?
Ideally you need a sample of pure oxidised protein and obtain the 278/597 ratio for that. Let's assume your final prep above (gel filtration step) is pure (ratio 0.85). (You may know whether this is true from SDS-page analysis?). Thus, any sample with a ratio of 0.85 must be pure. And a sample with value 1.7 is 50% pure (100*0.85/1.7), and a sample with a ratio of 8.5 is 10% pure, and so on.
Now, there are some assumptions/caveats here:
1. All proteins absorb at A278 similarly to plastocyanin
2. There is no error on the A278 & 597 measurements
Of course neither of these can ever be strictly true.
And you say that the 'oxidised' form absorbs at A597, but you do not mention the reduced form. If some protein is reduced, and especially if that form does not absorb at A597, then the method becomes unreliable and you will underestimate the purity, as the reduced form will contribute to A278 but not to A597. If you can deliberately oxidise the sample prior to determination of the ratio, then that would be fine.
Finally, just personal preference, I would do the ratio as 597/278 because this gives low values for homogenates and relatively high values for pure proteins, which just feels right. i.e. By analogy with enzymes, specific activity values (units per mg) increase as the purity increases. Mathematically, however, either way round is fine.
yes, we have oxidized our protein to make sure there are no reduced forms that are absorbing at 278. Thank you for your comprehensive answer.
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