HPLC monitoring of 45 kDa protein cleavage reaction

[Crevetus]

Hi everyone,

I'm looking for a reliable method to monitor (and quantitate) the cleavage reaction of a 45 kDa protein by a protease. The cleaved fragment is 1 kDa long, so the targeted analytes should have 45, 44 and 1 kDa. The uncleaved protein is hydrophilic and stable in HEPES buffer for several weeks. The SDS-PAGE analysis of the reaction mixture is not really conclusive and it takes very long. Do you have any suggestions for a system capable of performing the separation on a normal PDA-HPLC?

Thanks in advance!

[JMB]

Well, size- exclusion LC will separate 45 and 1 kDa, but not 44 & 45 kDa., unless the conformation changes significantly.
A lit search may turn up tricks for resolving v. Similar MW proteins. If you are fortunate, clipping off the decapeptide may change the relative hydrophilic nature of the 44/45 kDa components enough to enable a reversed phase method to resolve them.

Regards,
JMB

[ScottHorn]

I agree with the above post. You will NEVER be able to separate 44 kD from 45 kD using SEC. If you somehow manage that feat, I will buy several dozen of whatever column you used.

In addition to the above suggestion about a potential reversed phase approach, you could also consider an ion exchange based approach. If the part that you're cleaving contributes significantly to the overall charge of the protein, you may have luck with an ion exchange route. If you have the capability to do isoelectric focusing, you can get an idea of how well such a method would work, and even some starting points for method development.

Another idea that comes to mind, would be to monitor the amount of 1 kD material produced over time, perhaps by taking the reaction mixture and spinning it in a centrifugal concentrator with a suitable MWCO, say 10 kd or so. From there, you could measure UV absorbance of the filtrate vs the retained protein, and calculate how many moles of 1 kD material you have vs moles of starting material. This assumes you know the extinction coefficients of both.

[Crevetus]

Thanks for the suggestions. I have a Yarra SEC-2000 column and I will try to "catch" the 1 kDa rest.

ScottHorn, nice idea with the amicons. While it's unlikely that one can quantitate the reaction by it (the 1 kDa peptide will also stick to the membrane), at least it could be a good indicator about how well is the reaction doing.

Enjoy your day!