Ultrafiltration binding analyses of glycated albumin with a 3D-printed syringe attachment.
Authors of this article are:
Castiaux AD Pinger CW Spence DM.
A summary of the article is shown below:
Protein-ligand binding assays facilitate the understanding of biomolecular interactions. Classical equilibrium dialysis methods are often used for accurate determination of binding properties. While accurate, the long equilibration times associated with the technique (> 6 h) hinder throughput. Here, in an attempt to gather high-accuracy results while reducing total analysis time, a low pressure ultrafiltration method that relies on a simple membrane-containing syringe attachment was developed. A minimal portion (1-2%) of the solution containing the binding analytes of interest is driven through the membrane pores and collected for analysis. Specifically, the device was used to investigate the binding affinity between Zn2+ and either normal human serum albumin (nHSA) or a commercially purchased glycated human serum albumin (gHSA). Both of these ligand/protein-binding systems have implications in type 1 diabetes. The device was then used to investigate the binding between the various albumin types and C-peptide, the 31 amino acid peptide that is co-secreted with insulin from pancreatic β cells. Results for nHSA/Zn2+ binding obtained using the ultrafiltration method (Kd = 5.77 ± 0.19 × 10-7 M) were statistically equivalent with results reported using other methods. Importantly, the amount of Zn2+ bound to the nHSA was significantly different from the gHSA (97 ± 2% protein bound vs. 91 ± 3%, respectively p < 0.05). The binding affinity of C-peptide to nHSA (Kd = 2.4 ± 0.3 × 10-6 M) agreed with values reported in the literature using standard techniques. Unlike Zn2+ binding, the binding of C-peptide to nHSA was statistically equal to its binding to gHSA (77.7 ± 6.2 and 78.8 ± 7.4%, respectively), suggesting that C-peptide replacement therapy in people with T1D may be strongly dependent upon the characteristics of Zn2+ binding to human serum albumin. Graphical ᅟ.
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This article is a good source of information and a good way to become familiar with topics such as: Binding affinity constants;Bioanalytical methods;Metals/heavy metals;Microfluidics/microfabrication;Separations/instrumentation.
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