[10] Study of protein-protein and of protein-ligand interactions by potentiometric methods

Laskowski, M.; Finkenstadt, William R.

doi:10.1016/s0076-6879(72)26012-8

Cited by 34 publications

(26 citation statements)

References 24 publications

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“…I The rigorous description of eq 1 and derivation of eq 3 refer to a standard state in which all species are fully protonated and require the introduction of Ac as the number of new cationic groups created as a consequence of complex formation (Laskowski & Finkenstadt, 1972). We have chosen to simplify our description of the theory by not referring to this standard state so that Ac is implicit in our description of q. by deletion of N-and C-terminal sequence from the lipase form (Funk, 1990) and expressed in exactly the same expression system as used for the lipase-solubilized protein .…”

Section: Methodsmentioning

confidence: 99%

“…Lebowitz and Laskowski (1962) were the first to use potentiometric measurements for quantitative determination of the difference titration curve for a protein-protein complex and to relate it to the pH dependence of the association constant, KA. It can be shown (Edsall & Wyman, 1958;Laskowski & Finkenstadt, 1972;Wyman & Gill, 1990) that This equation can be integrated readily to yield which allows KA to be evaluated at any pH provided that KA is known at one reference pH and that the difference titration curve is known over the region between pH, and pH2. No specific information concerning the origins of the difference curve is required to make use of this relationship (Lebowitz & Laskowski, 1962;Rupley et al, 1967).…”

Section: Methodsmentioning

confidence: 99%

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Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions

Mr¹,

Pd²,

Ag³

1991

Biochemistry

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Two potentiometric methods have been used to study the pH-dependent changes in proton binding that accompany complex formation between cytochrome c and cytochrome b5. With one method, the number of protons bound or released upon addition of one cytochrome to the other has been measured as a function of pH. The results from these studies are correlated with the complexation-induced difference titration curve calculated from the titration curves of the preformed complex and of the individual proteins. Both methods demonstrate that complex formation at acid pH is accompanied by proton release, that complex formation at basic pH is accompanied by proton uptake, and that the change in proton binding at neutral pH, where stability of complex formation is maximal, is relatively small. Under all conditions studied, the stoichiometry of cytochrome c-cytochrome b5 complex formation is 1:1 with no evidence of higher order complex formation. Although the dependence of complex formation on pH for interaction between different species of cytochrome c and cytochrome b5 are qualitatively similar, they are quantitatively different. In particular, complex formation between yeast iso-1-cytochrome c and lipase-solubilized bovine cytochrome b5 occurs with a stability constant that is 10-fold greater than observed for the other two pairs of proteins under all conditions studied. Interaction between these two proteins is also significantly less dependent on ionic strength than observed for complexes formed by horse heart cytochrome c with either form of cytochrome b5.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions

Mr¹,

Pd²,

Ag³

1991

Biochemistry

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“…The change in proton binding that is associated with the binding of Zn 2+ , Co 2+ , Ni 2+ , Mn 2+ , Cu 2+ and Fe 3+ to apoFurC* (5.69-6.44 µM) was studied at 25 • C by the method of Laskowski and Finkenstadt [37] with a Radiometer ABU93 Triburet operated under computer control as described previously [38]. Difficulties related to oxidation of Fe 2+ by dissolved dioxygen prevented consideration of the binding of this species to FurC*.…”

Section: Potentiometric Titrationsmentioning

confidence: 99%

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

Bai

Rosell

Lige

et al. 2006

Biochemical Journal

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The functional properties of the recombinant C-terminal dimerization domain of the Pseudomonas aeruginosa Fur (ferric uptake regulator) protein expressed in and purified from Escherichia coli have been evaluated. Sedimentation velocity measurements demonstrate that this domain is dimeric, and the UV CD spectrum is consistent with a secondary structure similar to that observed for the corresponding region of the crystallographically characterized wild-type protein. The thermal stability of the domain as determined by CD spectroscopy decreases significantly as pH is increased and increases significantly as metal ions are added. Potentiometric titrations (pH 6.5) establish that the domain possesses a high-affinity and a low-affinity binding site for metal ions. The high-affinity (sensory) binding site demonstrates association constants (K(A)) of 10(+/-7)x10(6), 5.7(+/-3)x10(6), 2.0(+/-2)x10(6) and 2.0(+/-3)x10(4) M(-1) for Ni2+, Zn2+, Co2+ and Mn2+ respectively, while the low-affinity (structural) site exhibits association constants of 1.3(+/-2)x10(6), 3.2(+/-2)x10(4), 1.76(+/-1)x10(5) and 1.5(+/-2)x10(3) M(-1) respectively for the same metal ions (pH 6.5, 300 mM NaCl, 25 degrees C). The stability of metal ion binding to the sensory site follows the Irving-Williams order, while metal ion binding to the partial sensory site present in the domain does not. Fluorescence experiments indicate that the quenching resulting from binding of Co2+ is reversed by subsequent titration with Zn2+. We conclude that the domain is a reasonable model for many properties of the full-length protein and is amenable to some analyses that the limited solubility of the full-length protein prevents.

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“…In Table 2 we collect the relative Finally, note that a DH 0 of 010 kcal/mol was used review (19). Obviously, for a given pair of values of c a rapid one, to form the complex, followed by the slow isomerization of one of the reactants (20). We have thus chosen to work at pH 4.5, to be in the binding constant part of Fig.…”

Section: Calorimetrymentioning

confidence: 99%

Measurement of Biochemical Affinities with a Gill Titration Calorimeter

Harrous

Parody-Morreale

1997

Analytical Biochemistry

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[10] Study of protein-protein and of protein-ligand interactions by potentiometric methods

Cited by 34 publications

References 24 publications

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions

Proton linkage of complex formation between cytochrome c and cytochrome b5: electrostatic consequences of protein-protein interactions

Functional characterization of the dimerization domain of the ferric uptake regulator (Fur) of Pseudomonas aeruginosa

Measurement of Biochemical Affinities with a Gill Titration Calorimeter

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