Limitations in linearized analyses of binding equilibria: binding of TNP-ATP to the H4-H5 loop of Na/K-ATPase
Jazyk angličtina Země Německo Médium print-electronic
Typ dokumentu srovnávací studie, hodnotící studie, časopisecké články, práce podpořená grantem, validační studie
- MeSH
- adenosintrifosfát analogy a deriváty chemie MeSH
- algoritmy * MeSH
- chemické modely * MeSH
- fluorescenční barviva MeSH
- fluorescenční spektrometrie metody MeSH
- kinetika MeSH
- lineární modely * MeSH
- počítačová simulace MeSH
- reprodukovatelnost výsledků MeSH
- řízení kvality MeSH
- sekundární struktura proteinů MeSH
- senzitivita a specificita MeSH
- sodíko-draslíková ATPasa chemie MeSH
- vazba proteinů * MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- validační studie MeSH
- Názvy látek
- 2',3'-O-(2,4,6-trinitro-cyclohexadienylidine)adenosine 5'-triphosphate MeSH Prohlížeč
- adenosintrifosfát MeSH
- fluorescenční barviva MeSH
- sodíko-draslíková ATPasa MeSH
Binding of TNP-ATP [2',3'- O-(2,4,6-trinitrophenyl)adenosine 5'-triphosphate, a fluorescent analogue of ATP] to the K605 protein was studied. This is an isolated N-domain in the cytoplasmic loop of the Na/K-ATPase alpha-subunit, lying between membrane-spanning segments 4 and 5 (sequence L(354)-I(604)). A titration equation is derived that explicitly makes it possible to relate the fluorescence of TNP-ATP and K605 solutions to total probe concentration in the sample. Using this, it is possible to obtain the value of the dissociation constant from the titration experiment without resorting to the Scatchard plot, which is far from optimal from the statistical point of view. Using the new formula with non-linear regression analysis, a value of the dissociation constant K(D) for TNP-ATP binding to the K605 protein of 3.03 +/- 0.28 microM at 22 degrees C was obtained. A series of fits to simulated data with added noise demonstrated clearly the advantage of non-linear regression using the new formula over the commonly employed linear regression using the Scatchard plot. The procedure presented is generally applicable to binding assays using changes in the fluorescence of ligands on binding.
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