I would like to ask if there is calcium really unnecessary for thrombin activity ? I've found one publication, where they claimed that thrombin is Ca-dependent enzyme ... but there was no reference to this statement. I need to use thrombin to cut-off GST from my fusion protein, but my protein-of-interest is vulnerable to Ca and Ca lowest it's activity.
Here is the quote from one paper that mess my mind:
We have found that recombinant TGase loses activity in a matter
of hours in the presence of calcium, so we were concerned that
the calcium-dependent thrombin-mediated cleavage of Lai’s GSThTG2
fusion protein would lead to significant loss of activity of
the resulting hTG2.
First, you can look up enzyme properties, assay conditions, and lots of other info using the BRENDA database -- a great resource. www.brenda-enzymes.org
You should have sufficient activity by using a relatively high concentration of phosphate buffer around pH 7.5 - 8.0, without having to add extra calcium. I'm having trouble finding the reference that I'm thinking of -- but BRENDA should have lots of assay condition information for you.
I fully agree with Omid, thrombin loses its main calcium-binding sites upon activation.
Calcium is necessary for the activity of complexes which participate in thrombin generation but not for its activity.
and I also fully agree with my colleagues, "thrombin loses its main calcium-binding sites upon activation", but thrombin remains contaminated a residual Ca++, and it should be considered or purified thrombin from Ca++.
Thrombin does not need calcium for the cleavage of fibrinogen to fibrin.
Calcium is unnecesary for the cleavage of fibrinopeptide A and B from fibrinogen. You can try with some snake venom that work like thrombin.
The FIFTA is a new method to analyze Fibrinogen function in citrated plasma. Thrombin is added without recalcification and it works without problems even in Routine hospital analysis.
In our clotting assays thrombin works fine (meaning converts fibrinogen to fibrin) without any calcium.
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