A novel 3D-printed centrifugal ultrafiltration method reveals in vivo glycation of human serum albumin decreases its binding affinity for zinc.
A novel 3D-printed centrifugal ultrafiltration method reveals in vivo glycation of human serum albumin decreases its binding affinity for zinc.
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DOI：
10.1039/d0mt00123f
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发表时间：
2020-07-22
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血浆蛋白在体内被糖尿病患者血液中的高葡萄糖条件共价修饰，导致结构和功能的变化。人血清白蛋白（Human Serum Albumin， HSA）在血液中作为一种载体蛋白，结合多种配体并严格调节其生物利用度。已知HSA通过美拉德反应与葡萄糖反应，对其结合和递送某些配体（如金属）的能力产生不利影响。在这里，体内糖基化HSA与锌（Zn2+）之间的结合是通过使用3d打印设备开发的新型离心超滤方法来确定的。该方法快速（90分钟），能够进行高通量测量（24个样品），成本低（每个设备< 1.00美元），并且与其他结合技术相比，需要更少的样品体积（200 μL）。该装置用于测定Zn2+与商业上获得的糖基化水平为11.5%的正常HSA （nHSA）之间的平衡解离常数（Kd = 2.1（±0.5）× 10−7 M）。nHSA样品的糖基化部分被富集（gHSA, 65.5%），并使用硼酸亲和层析分离，发现与nHSA样品相比，Zn2+结合亲和力降低了2.3倍（Kd = 4.8（±0.8）× 10−7 M）。采用质谱法测定对照血浆（13.0%±0.8,n=3名供者）和糖尿病患者血浆（26.9%±6.6,n=5名供者）HSA糖化水平。此外，从1型糖尿病患者的血浆中分离出HSA，发现Zn2+的糖基化水平为24.1%,Kd = 3.3（±0.5）× 10−7 M，与nHSA相比，HSA的结合亲和力降低了1.5倍。这些发现表明，糖基化HSA水平的升高导致与Zn2+结合减少，这可能与糖尿病相关的并发症有关。
Plasma proteins are covalently modified in vivo by the high-glucose conditions in the bloodstreams of people with diabetes, resulting in changes to both structure and function. Human Serum Albumin (HSA) functions as a carrier-protein in the bloodstream, binding various ligands and tightly regulating their bioavailability. HSA is known to react with glucose via the Maillard reaction, causing adverse effects on its ability to bind and deliver certain ligands, such as metals. Here, the binding between in vivo glycated HSA and zinc (Zn2+) was determined using a novel centrifugal ultrafiltration method that was developed using a 3D-printed device. This method is rapid (90 minutes), capable of high-throughput measurements (24 samples), low-cost (<$1.00 USD per device) and requires lower sample volumes (200 μL) compared to other binding techniques. This device was used to determine an equilibrium dissociation constant between Zn2+ and a commercially obtained normal HSA (nHSA) with a glycation level of 11.5% (Kd = 2.1 (± 0.5) × 10−7 M). A glycated fraction of the nHSA sample was enriched (gHSA, 65.5%) and isolated using boronate-affinity chromatography, and found to have a 2.3-fold decrease in Zn2+ binding-affinity (Kd = 4.8 (± 0.8) × 10−7 M) when compared to the nHSA sample. The level of glycation of HSA in control plasma (13.0% ± 0.8, n=3 donors) and plasma from people with diabetes (26.9% ± 6.6, n=5 donors) was assessed using mass spectrometry. Furthermore, HSA was isolated from plasma obtained in-house from a person with type 1 diabetes and found to have a glycation level of 24.1% and Kd = 3.3 (± 0.5) × 10−7 M for Zn2+, revealing a 1.5-fold decrease in binding affinity compared to nHSA. These findings suggest that increased levels of glycated HSA result in reduced binding to Zn2+, which may have implications in complications associated with diabetes.
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DOI：
10.1016/j.cca.2013.07.013
发表时间：
2013-10-21
期刊：
Clinica chimica acta; international journal of clinical chemistry
影响因子：
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作者：
Anguizola J;Matsuda R;Barnaby OS;Hoy KS;Wa C;DeBolt E;Koke M;Hage DS
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Hage DS
DOI：
10.7762/cnr.2018.7.4.229
发表时间：
2018-10
期刊：
Clinical nutrition research
影响因子：
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Yang SJ
DOI：
10.1016/j.bbalip.2018.09.007
发表时间：
2019-04
期刊：
Biochimica et biophysica acta. Molecular and cell biology of lipids
影响因子：
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作者：
Coverdale JPC;Khazaipoul S;Arya S;Stewart AJ;Blindauer CA
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Blindauer CA
影响因子：
8.4
作者：
Handing KB;Shabalin IG;Kassaar O;Khazaipoul S;Blindauer CA;Stewart AJ;Chruszcz M;Minor W
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Minor W
DOI：
10.1111/jth.12771
发表时间：
2015-01
期刊：
Journal of thrombosis and haemostasis : JTH
影响因子：
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作者：
Kassaar O;Schwarz-Linek U;Blindauer CA;Stewart AJ
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Stewart AJ