Determination of Equilibrium Constants of Glibenclamide-Human Serum Albumin Interactions by High-Performance Affinity Chromatography

Sophia How Author

07/28/2020 Added

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The interaction between glibenclamide and HSA in normal and modified form of proteins.

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[00:00:00.000]Hi everyone. My name is Sophia How.
[00:00:02.190]And I'll be presenting my project on
[00:00:04.270]Determination of Equilibrium Constant of
[00:00:06.619]Glibenclamide- Human Serum Albumin
[00:00:08.940]Interaction by High-Performance Affinity
[00:00:10.624]Chromatography.
[00:00:11.803]In 2018, approximately 10.5% of the
[00:00:14.869]population, or 34.2 million people in the
[00:00:18.229]United States were diagnosed with Type II
[00:00:20.151]diabetes. Type II diabetes is a metabolic
[00:00:22.426]disorder in which the pancreas does not
[00:00:24.876]produce enough insulin. It is most common
[00:00:27.818]form of diabetes, affecting 90-95% of the
[00:00:32.068]patients who have the condition.
[00:00:34.098]Human Serum Albumin as HSA is the most
[00:00:37.581]abundant transport protein with two major
[00:00:40.566]drug binding sites shown in Figure 1 that
[00:00:42.912]demonstrate the two sites- Sudlow site 1
[00:00:45.741]and 2 which R-warfarin and L-tryptophan
[00:00:53.071]will bind with the HSA.
[00:00:54.818]The glycation process in Figure 2
[00:00:57.603]is when glucose and protein bind together
[00:01:01.603]to form a Schiff’s base and it rearranged
[00:01:06.013]to a more stable product- Amadori product.
[00:01:08.927]Then it will undergoes oxidation,
[00:01:12.467]dehydration or degradation to get Advanced
[00:01:17.758]Glycation End-product as AGE modified HSA.
[00:01:24.248]HSA in normal and modified forms will
[00:01:27.558]bind with Glibenclamide, the drug shown in
[00:01:31.238]Figure 3. It is a drug that treats with
[00:01:34.551]Type II diabetes.
[00:01:37.653]The goal of this study is to examine the
[00:01:40.103]interaction of Glibenclamide with HSA in
[00:01:44.178]its normal and various modified forms
[00:01:47.916](methylglyoxal-AGE and glyoxal-AGE) of the
[00:01:51.662]protein. The method shown in Figure 4,
[00:01:56.592]shows the sample runs through the High
[00:01:59.152]Performance Affinity Chromatography
[00:02:02.594]as HPAC the sample will bind to the
[00:02:07.470]binding agents and leftover with non-
[00:02:11.470]retained sample will leave out. We will
[00:02:15.871]be injecting the drug solution onto 10
[00:02:19.871]millimters times 2.1 millimeters affinity
[00:02:24.231]microcolumns. And the experiment were
[00:02:26.918]carried at the flow rate of 0.5mL/min and
[00:02:30.918]the temperature of 37 degree Celsius.
[00:02:38.260]The R-warfarin and L-tryptophan were used
[00:02:41.918]as a site-specific probe to examine the
[00:02:44.418]HSA and its modified forms.
[00:02:47.767]And we will be performing eight different
[00:02:50.307]mobile phase concentrations from 0
[00:02:52.686]micromolar to 20 micromolars.
[00:02:58.686]The result shown in Figure 5 is that the
[00:03:05.709]retention time on the x-axis versus the
[00:03:09.050]absorbance of the probe being measured in
[00:03:11.730]the interaction of glibenclamide and
[00:03:13.921]l-tryptophan with concentrations ranging
[00:03:18.150]from 0 micromolar to 20 micromolars.
[00:03:22.597]Using Figure 5 we are able to plot onto
[00:03:25.847]Figure 6 with various concentrations of
[00:03:32.008]Glibenclamide on the x-axis vs 1over the
[00:03:35.938]retention factor and using Equation 1 we
[00:03:39.619]can calculate its equilibrium constant in
[00:03:44.805]HSA modified- AGEs- control and diabetic
[00:03:48.655](methylglyoxal and glyoxal) shown in
[00:03:52.511]Table 1.
[00:03:55.373]In table 1, the normal HSA has a greater
[00:03:58.247]binding constant when compared to control
[00:04:01.468]diabetic glyoxal and control diabetic
[00:04:05.127]methylglyoxal. In Figure 7, we see the
[00:04:10.137]decrease when comparing any of the HSA
[00:04:15.627]modified forms back to normal HSA.
[00:04:20.658]The conclusion overall of this experiment
[00:04:23.715]is the high performance affinity
[00:04:25.766]chromatography zonal competition studies
[00:04:28.191]were used to examine the interaction of
[00:04:30.663]Glibenclamide binding with normal and AGE-
[00:04:35.520]modified HSA at Sudlow sites I and II.
[00:04:39.178]The global affinity constants for the
[00:04:42.764]interaction of Glibenclamide with normal
[00:04:45.163]HSA and AGE-modified HSA are calculated
[00:04:49.263]using Equation 1 and shown in Table 1.
[00:04:56.556]There was an overall 0.25 to 0.39 fold
[00:05:01.026]changes when comparing normal HSA
[00:05:03.766]constants to one of the modified forms
[00:05:07.326]of the protein, as shown in Figure 7.
[00:05:11.500]This study demonstrated that drug-protein
[00:05:14.110]interactions can be used to estimate
[00:05:16.355]binding constants in a relatively fast
[00:05:19.073]and reproducible way.
[00:05:21.144]I would like to acknowledge the
[00:05:25.144]Department of Chemistry at University
[00:05:28.039]of Nebraska-Lincoln and Queensborough
[00:05:30.319]Community College. And I also like to
[00:05:33.200]acknowledge Dr. Mark Griep and Dr. David
[00:05:36.830]Hage and also Dr. Paris Svoronos and the
[00:05:41.791]National Science Foundation Research
[00:05:44.667]Experience for Undergraduates. Thank you
[00:05:47.999]for listening.

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Determination of Equilibrium Constants of Glibenclamide-Human Serum Albumin Interactions by High-Performance Affinity Chromatography

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