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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
Allosterically coupled multi-site binding of T to human serum albumin
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<blockquote data-quote="madman" data-source="post: 190001" data-attributes="member: 13851"><p><strong>Figure 3: <span style="color: rgb(184, 49, 47)">Fluorescence spectroscopy analysis provides direct evidence for multiple binding sites and allostery.</span> testosterone was titrated into a solution of 1 µM HSA in a single quartz cuvette and the system was excited at 295 nm to target tryptophan residues. As shown in <span style="color: rgb(184, 49, 47)">Figure 3A, </span>titration of testosterone into HSA: OA mixture, led to quenching of fluorescence emission from tryptophan residues. Testosterone was sequentially increased from 0 to 60 µM and at each concentration, the total delta intensity <span style="color: rgb(184, 49, 47)">(defined in the text) </span>was calculated and plotted as a function of [T] <span style="color: rgb(184, 49, 47)">(Figure 3B)</span>. The data were then fit with three distinct binding models: <span style="color: rgb(184, 49, 47)">(i)</span> the extent linear model assuming 1:1 binding stoichiometry for HSA: T complex formation <span style="color: rgb(184, 49, 47)">(Figure 3B, red curve)</span>. The unconstrained best fit to this model found a Kd of 528 nM, but it is clear that it does not adequately describe the data. <span style="color: rgb(184, 49, 47)">(ii) </span>Two, non-interacting, independent binding sites with distinct Kds. <span style="color: rgb(44, 130, 201)">Figure 3B, the blue curve</span> shows the best fit to the two-site specific binding model while constraining the Bmax value to one-half of the delta intensities (in the absence of relative quantum yield for the intermediate binding states, the two Bmax values were constrained to 1.8 × 106, the midpoint between the minimum and maximum Σ∆I values. The model with two independent sites also poorly fit <span style="color: rgb(184, 49, 47)">(two discrete Kd values - 81.9 nM and 18.1 µM)</span> the data.<span style="color: rgb(184, 49, 47)"> (iii) </span>Two interacting binding sites <span style="color: rgb(26, 188, 156)">(Figure 3B, green curve)</span>. The data fits resulted in the determination of two interacting sites, exhibiting a high-affinity Kd of 17.8 nM and a low-affinity Kd of 12.3 µM. <span style="color: rgb(184, 49, 47)">Figure 3C</span> shows changes in emission from bis-ANS repartitioning upon titration of testosterone from 0 to 1 mM into µM HSA incubated with 10 µM bis-ANS.<span style="color: rgb(184, 49, 47)"> Figure 3C, the red curve</span> shows the fits to two-site specific binding model fit using the Kd constraints obtained from the fit of the steady-state tryptophan emission data from the <span style="color: rgb(26, 188, 156)">green curve in Figure 3B.</span> Collectively, these independent measures of testosterone-induced perturbations in fluorescence emission from intrinsic <span style="color: rgb(184, 49, 47)">(tryptophan)</span> and extrinsic <span style="color: rgb(184, 49, 47)">(bis-ANS) </span>probes provide evidence of multiple, interacting testosterone binding sites on HSA.</strong></p><p>[ATTACH=full]11380[/ATTACH]</p></blockquote><p></p>
[QUOTE="madman, post: 190001, member: 13851"] [B]Figure 3: [COLOR=rgb(184, 49, 47)]Fluorescence spectroscopy analysis provides direct evidence for multiple binding sites and allostery.[/COLOR] testosterone was titrated into a solution of 1 µM HSA in a single quartz cuvette and the system was excited at 295 nm to target tryptophan residues. As shown in [COLOR=rgb(184, 49, 47)]Figure 3A, [/COLOR]titration of testosterone into HSA: OA mixture, led to quenching of fluorescence emission from tryptophan residues. Testosterone was sequentially increased from 0 to 60 µM and at each concentration, the total delta intensity [COLOR=rgb(184, 49, 47)](defined in the text) [/COLOR]was calculated and plotted as a function of [T] [COLOR=rgb(184, 49, 47)](Figure 3B)[/COLOR]. The data were then fit with three distinct binding models: [COLOR=rgb(184, 49, 47)](i)[/COLOR] the extent linear model assuming 1:1 binding stoichiometry for HSA: T complex formation [COLOR=rgb(184, 49, 47)](Figure 3B, red curve)[/COLOR]. The unconstrained best fit to this model found a Kd of 528 nM, but it is clear that it does not adequately describe the data. [COLOR=rgb(184, 49, 47)](ii) [/COLOR]Two, non-interacting, independent binding sites with distinct Kds. [COLOR=rgb(44, 130, 201)]Figure 3B, the blue curve[/COLOR] shows the best fit to the two-site specific binding model while constraining the Bmax value to one-half of the delta intensities (in the absence of relative quantum yield for the intermediate binding states, the two Bmax values were constrained to 1.8 × 106, the midpoint between the minimum and maximum Σ∆I values. The model with two independent sites also poorly fit [COLOR=rgb(184, 49, 47)](two discrete Kd values - 81.9 nM and 18.1 µM)[/COLOR] the data.[COLOR=rgb(184, 49, 47)] (iii) [/COLOR]Two interacting binding sites [COLOR=rgb(26, 188, 156)](Figure 3B, green curve)[/COLOR]. The data fits resulted in the determination of two interacting sites, exhibiting a high-affinity Kd of 17.8 nM and a low-affinity Kd of 12.3 µM. [COLOR=rgb(184, 49, 47)]Figure 3C[/COLOR] shows changes in emission from bis-ANS repartitioning upon titration of testosterone from 0 to 1 mM into µM HSA incubated with 10 µM bis-ANS.[COLOR=rgb(184, 49, 47)] Figure 3C, the red curve[/COLOR] shows the fits to two-site specific binding model fit using the Kd constraints obtained from the fit of the steady-state tryptophan emission data from the [COLOR=rgb(26, 188, 156)]green curve in Figure 3B.[/COLOR] Collectively, these independent measures of testosterone-induced perturbations in fluorescence emission from intrinsic [COLOR=rgb(184, 49, 47)](tryptophan)[/COLOR] and extrinsic [COLOR=rgb(184, 49, 47)](bis-ANS) [/COLOR]probes provide evidence of multiple, interacting testosterone binding sites on HSA.[/B] [ATTACH type="full"]11380[/ATTACH] [/QUOTE]
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Testosterone Replacement, Low T, HCG, & Beyond
Testosterone and Men's Health Articles
Allosterically coupled multi-site binding of T to human serum albumin
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