Validation of SLOMO using model biomolecular interactions with
known affinities (a–d) Van–AcAA, (e–h) PT–STI,
and (i–l) BLG–BLG. (a) Representative ESI mass spectra
acquired in the positive ion mode at three different times for a mixture
of ammonium acetate (200 mM, pH 6.9) solutions: solution 1, Van (2
μM) and AcAA (10 μM); solution 2, Van (2 μM) and
AcAA (500 μM). (b) Plot of time-dependent ΔRapp values. The inset shows ΔRapp values measured at early times, and the dashed line (blue)
indicates two standard deviations (2δ) from the mean. (c) Plot
of time-dependent relative response factors (RFVan/(Van+AcAA)) measured for Van and the Van–AcAA complex. (d) Plot of the
fraction of Van bound to AcAA versus the initial AcAA concentration
determined without (black circles) and with (red circles) consideration
of RFVan/(Van+AcAA). Solid curves represent the best fit
of eq 11 to the experimental
data. (e) Representative ESI mass spectra acquired in the positive
ion mode at three different times for a mixture of ammonium acetate
(200 mM, pH 4.5) solutions: solution 1, PT (3 μM) and STI (0.5
μM); solution 2, PT (3 μM) and STI (30 μM). (f)
Plot of time-dependent ΔRapp values.
The inset shows ΔRapp values measured
at early mixing times. (g) Plot of time-dependent relative response
factors (RFPT/(PT+STI)) measured for PT and the PT–STI
complex. (h) Plot of the fraction of PT bound to STI versus the initial
STI concentration determined without (black circles) and with (red
circles) consideration of RFPT/(PT+STI). Solid curves represent
the best fit of eq 11 to the experimental data. (i) Representative ESI mass spectra acquired
in the positive ion mode at three different times for a mixture of
ammonium acetate (200 mM) solutions of BLG (3 μM): solution
1, pH 6.9; solution 2, pH 10.6. (j) Plot of time-dependent ΔRapp values. (k) Plot of time-dependent relative
response factors (RFBLG/(BLG)2) measured for
the BLG monomer and dimer. (l) Plot of the fraction of BLG present
as the dimer versus the initial BLG (monomer) concentration (1–9
μM) measured without (black circles) and with (red circles)
consideration of RFM/D. Solid curves represent the best
fit of eq 17 to the
experimental data.