DOI: 10.5281/zenodo.10881688
title: Protein sialylation affects the pH-dependent binding of ferric ion to human serum transferrin - experimental data

description: Experimental data for a paper entitled Protein sialylation affects the pH-dependent binding of ferric ion to human serum transferrin (https://doi.org/10.1039/D4DT01311E).

 

Separate files are provided with the following information:

 

1. Experimental data:

a) "Fluorescence + IFE corrections" worksheet contains the fluorescence data used for the subsequent calculations.

Fluorescence values were measured in triplicate the Tecan Spark M10 multimode microplate reader using multiple z-positions (distance from the lens to the bottom of the microplate well) and baseline correction. 

Fluorescence was corrected for the inner filter effect and normalized as described in the Supplementary Information (citation data will be updated pending publication).

Additional information on the z-position inner filter effect correction method (ZINFE) can be found here: https://doi.org/10.1021/acs.analchem.2c01031.

b) "Absorbance spectra" worksheet contains the averaged absorbance spectra and absorbance spectra of each replicated measurement.

 

2. Titrations

The dependence of the measured fluorescence on the concentration of added FeNTA was modelled according to the binding polynomial for two iron binding sites (https://doi.org/10.1016/S0076-6879(08)04205-5).

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 (https://doi.org/10.1039/d3ay01261a), the least squares calculation of the fluorescence according to the binding polynomial for each data point using the solver routine in Excel, and the “jackknife” procedure for estimating the uncertainties of the titration parameters (https://doi.org/10.1021/ed075p119). 

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.

 

3. Results and sensitivity analyses:

Local sensitivity analysis was performed to assess the impact of concentration fluctuations on the uncertainties associated with the results.

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.

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.

Standard deviations of the obtained titration parameters were adjusted using this expanded set.

 

4. All data used for tables and plots in the manuscript, including statistical analyses and miscellaneous data:

a) "Tables" worksheet contains all tables in the manuscript and the Supplementary Information (citation data will be updated pending publication),

b) "fsp" worksheet contains the data on the pH-dependence of the site preference factor, fsp (https://doi.org/10.1042/bj1930717),

c) "ΔΔrG" worksheet contains all ΔΔrG values reported in the manuscript,

d) "Error propagation" contains the error propagation calculations for all values reported in the manuscript.

 
description: This work was supported by funding from the Croatian Science Foundation grant UIP-2017-05-9537 – Glycosylation as a factor in the iron transport mechanism of human serum transferrin (GlyMech). Valentina Borko was additionally financed by the Croatian Science Foundation grant DOK-2018-09-1042. Additional support was provided by the European Regional Development Fund grants for 'Strengthening of Scientific Research and Innovation Capacities of the Faculty of Pharmacy and Biochemistry at the University of Zagreb' (KK.01.1.1.02.0021), 'Development of methods for production and labelling of glycan standards for molecular diagnostics' (KK.01.1.1.07.0055) and 'Scientific center of excellence for personalized health care' (KK.01.1.1.01.0010). The N-glycan profiling by UHPLC-MS was provided by the Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Croatia.