Low-pressure chromatographic separation and UV/Vis spectrophotometric characterization of the native and desialylated human apo-transferrin

Abstract

Low-pressure pH gradient ion exchange separation provides a fast, simple and cost-effective method for preparative purification of native and desialylated apo-transferrin. The method enables easy monitoring of the extent of the desialylation reaction and also the efficient separation and purification of protein fractions after desialylation. The N -glycan analysis shows that the modified desialylation protocol successfully reduces the content of the sialylated fractions relative to the native apo-transferrin. In the optimized protocol, the desialylation capacity is increased by 150 %, compared to the original protocol provided by the manufacturer. The molar absorption coefficients in the near-UV region for the native and desialylated apo-transferrin differ by several percent, suggesting a subtle dependence of the glycoprotein absorbance on the variable sialic acid content. The method can easily be modified for other glycoproteins and is particularly appropriate for quick testing of sialic acid content in the protein glycosylation patterns prior to further verification by mass spectrometry.

Influence of Desialylation on the Drug Binding Affinity of Human Alpha-1-Acid Glycoprotein Assessed by Microscale Thermophoresis

“…To quantify sialic acid content in native and desialylated AAG, the index of sialylation ( IS ) was used. The IS value was calculated using Equation (5): where n represents the number of N-glycan fractions, f i is the percentage of a specific N-glycan fraction of the total assigned N-glycans, and s i is the number of sialic acids present in the structure of each N-glycan fraction [ 44 ].…”

“…In our previous study, we demonstrated that the absorbance of native human transferrin exhibits no significant dependence on changes in ionic strength up to 1.5 M [44].…”

“…where n represents the number of N-glycan fractions, f i is the percentage of a specific N-glycan fraction of the total assigned N-glycans, and s i is the number of sialic acids present in the structure of each N-glycan fraction [44]. To quantify sialic acid content in native and desialylated AAG, the index of sialylation (IS) was used.…”

“…where n represents the number of N-glycan fractions, fi is the percentage of a specific Nglycan fraction of the total assigned N-glycans, and si is the number of sialic acids present in the structure of each N-glycan fraction [44]. The calculated IS values for native and desialylated AAG samples are approximately 270 and 5, respectively, indicating over 98% reduction in the sialic acid content for the enzymatically desialylated AAG.…”

Preparation and characterization of iron(iii) nitrilotriacetate complex in aqueous solutions for quantitative protein binding experiments

“…This coefficient was determined using the modied Edelhoch method, 44 and the result is consistent with previously published values (84.0 × 10 3 M −1 cm −1 and 84.8 × 10 3 M −1 cm −1 ). 45,46 The titration included 31 data points with separate preparations. In each case, an aliquot of FeNTA solution was added to a 0.1 mM human serum transferrin (hTf) solution.…”