Hello. This year we started using ITC in a bit unconventional way to study enzyme kinetics of beta-lactamase (there is already literature data that showed it works). The principle is fairly similiar to binding experiments, the difference is that after every injection of substrate to enzyme the baseline does not return to zero, but there is a slight displacement. This differences can than be converted to reaction rates if you know the ∆H of the reaction. The biggest problem we are facing is huge inconsistency in data, especially with controls, where we titrate just substrate to buffer (see the pictures). Also, at the beginning of each titration (both enzyme and controls) we get this endothermic dips, which is weird, because dilution heat should produce exotermic peaks, which are clearly showing up after couple of injections. Anyone has clue what might be going on or have some practical advice? Some useful experimental information: we use Affinity ITC, 190 ul sample cell, 30x2 ul injections with 100s spacing.
Perhaps the reason is that the substrate self-associates when at high concentration, so that in addition to the heat of dilution, there is also an effect of dissociation of the complex/aggregate upon dilution, which may be endothermic.
Thanks for the feedback Adam. We observed this for both substrates we used, ampicillin and carbenicillin. The concentrations of substrate in the injection syringe is 1 mM. Do you think that both antibiotics are prone to aggregation at this concentration?
I found papers on self-association of ampicillin and other penicillins, although the concentration range mentioned is much higher than 1 mM.
Article A study of the behavior of ampicillin in aqueous solution an...
https://d1wqtxts1xzle7.cloudfront.net/51863244/Colloidal_properties_of_benzylpenicillin20170219-4054-120iflx-libre.pdf?1487534326=&response-content-disposition=inline%3B+filename%3DColloidal_properties_of_benzylpenicillin.pdf&Expires=1694213746&Signature=E1~waQzXwbU8UNB~qf7PrSOOhk5DMmlnxDVh-lUmLAL2hybgmoJ5SE4G~elrB5hFaq8QeDTgG8h941noJARP0-u9EdBkbTMp1v7nSu~-1~KRS12IP1W6rUqbb9tZneeQd7fPd11K7YoMizktSdnuag3TLVA8YP4O3HBYcdyI59n3YmmKj1Hp306~cvAU0GX3qICEmDf2t5czNF2z004FDeFx~oAbMNqIaiuy3lxY7ZnvTbO~62t1uyPNcTTWPN0n3eS7YZZpwc-qDvTdrJSRDa~31W~MqFCRS0LEIlz9LXlIouUpb-5SoECGYDmFDArcvFgmx~GyZ6nA~qf2bgnVBg__&Key-Pair-Id=APKAJLOHF5GGSLRBV4ZA
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