Protein sialylation affects the pH-dependent binding of ferric ion to human serum transferrin - experimental data

<p>Experimental data for a paper entitled&nbsp;<em>Protein sialylation affects the pH-dependent binding of ferric ion to human serum transferrin</em> (<a href="https://doi.org/10.1039/D4DT01311E">https://doi.org/10.1039/D4DT01311E</a>).</p> <p>&nbsp;</p> <p>Separate files are provided with the following information:</p> <p>&nbsp;</p> <p>1. Experimental data:</p> <p>a) "Fluorescence + IFE corrections" worksheet contains the fluorescence data used for the subsequent calculations.</p> <p>Fluorescence values were measured in triplicate the Tecan Spark M10 multimode microplate reader using multiple&nbsp;<em>z</em>-positions (distance from the lens to the bottom of the microplate well) and baseline correction.&nbsp;</p> <p>Fluorescence was corrected for the inner filter effect and normalized as described in the Supplementary Information (citation data will be updated pending publication).</p> <p>Additional information on the <em>z</em>-position inner filter effect correction method (ZINFE) can be found here: <a title="DOI URL" href="https://doi.org/10.1021/acs.analchem.2c01031" target="_blank" rel="noopener">https://doi.org/10.1021/acs.analchem.2c01031</a>.</p> <p>b) "Absorbance spectra" worksheet contains the averaged absorbance spectra and absorbance spectra of each replicated measurement.</p> <p>&nbsp;</p> <p>2. Titrations</p> <p>The dependence of the measured fluorescence on the concentration of added FeNTA was modelled according to the binding polynomial for two iron binding sites (<a href="https://doi.org/10.1016/S0076-6879(08)04205-5" target="_blank" rel="noopener">https://doi.org/10.1016/S0076-6879(08)04205-5</a>).</p> <p>The results for all titrations are provided as separate files with 4 worksheets each: titration data, calculation of the free iron according to the binding polynomial (<a href="https://doi.org/10.1039/d3ay01261a" target="_blank" rel="noopener">https://doi.org/10.1039/d3ay01261a</a>), the least squares calculation of the fluorescence according to the binding polynomial for each data point using the solver routine in Excel, and the &ldquo;jackknife&rdquo; procedure for estimating the uncertainties of the titration parameters (<a href="https://doi.org/10.1021/ed075p119" target="_blank" rel="noopener">https://doi.org/10.1021/ed075p119</a>).&nbsp;</p> <p>Separate files are provided for the determination of the active protein fraction and the fluorescence signal corresponding to the complete saturation of the binding sites.</p> <p>&nbsp;</p> <p>3. Results and sensitivity analyses:</p> <p>Local sensitivity analysis was performed to assess the impact of concentration fluctuations on the uncertainties associated with the results.</p> <p>Concentrations were adjusted to both higher (H) and lower (L) estimates, based on adding or subtracting one standard deviation from the average concentrations, creating four scenarios: LL, LH, HL, HH.</p> <p>The results for all titrations are provided as separate files with 5 worksheets each: the results obtained directly from titration data and the results obtained by all 4 scenarios.</p> <p>Standard deviations of the obtained titration parameters were adjusted using this expanded set.</p> <p>&nbsp;</p> <p>4. All data used for tables and plots in the manuscript, including statistical analyses and miscellaneous data:</p> <p>a) "Tables" worksheet contains all tables in the manuscript and the Supplementary Information (citation data will be updated pending publication),</p> <p>b) "fsp" worksheet contains the data on the pH-dependence of the site preference factor, <em>f</em>sp (<a href="https://doi.org/10.1042/bj1930717" target="_blank" rel="noopener">https://doi.org/10.1042/bj1930717</a>),</p> <p>c) "&Delta;&Delta;rG" worksheet contains all &Delta;&Delta;r<em>G</em> values reported in the manuscript,</p> <p>d) "Error propagation" contains the error propagation calculations for all values reported in the manuscript.</p> <p>&nbsp;</p>