Analytical and Bioanalytical Chemistry Electronic Supplementary Material Analysis of glipizide binding to normal and glycated human serum Albumin by high-performance affinity chromatography Ryan Matsuda, Zhao Li, Xiwei Zheng, David S. Hage
Supplementary Material Analysis of Frontal Analysis Data According to Eq. (5) Frontal analysis data for the binding of glipizide with normal HSA and glycated HSA were also examined by using plots produced according to Eqs. (2) or (4). A typical doublereciprocal plot of 1/m Lapp vs. 1/[A] for such as a system is given in Fig. S1 (see following page). As is shown in this figure, a linear response was noted at low concentrations of glipizide, or high 1/[Glipizide]. However, at higher concentrations, or lower values for 1/[Glipizide], deviations from the linear response was observed. This deviation confirmed that glipizide was interacted at more than one type of site on normal HSA or glycated HSA. The linear region that was noted at the higher values of 1/[Glipizide] were used with Eq. (5) to provide an estimate for the association equilibrium constant for the high affinity sites of glipizide on these proteins. In Fig. S1, the best-fit line over this linear region gave a correlation coefficient of 0.9971 (n = 6) and an estimated association equilibrium constant of 1.1 (± 0.1) 10 5 M -1. Similar linear fits to the lower concentration data were observed for ghsa1 and ghsa2, which gave correlation coefficients of 0.9994 (n = 6) and 0.9992 (n = 6), respectively. The corresponding association equilibrium constants that were estimated for the high affinity sites were 1.1 (± 0.1) 10 5 M -1 for ghsa1 and 1.2 (± 0.1) 10 5 M -1 for ghsa2. All of the values were statistically identical to each other at the same at the 95% confidence interval. Zonal Elution Competition Studies at Sudlow Site I The linear region obtained in Fig. 5(a) for the normal HSA column at low glipizide concentrations had a correlation coefficient of 0.9999 (n = 4). The ratio of the slope and intercept for this linear region gave an association equilibrium constant of 6.1 (± 0.1) 10 4 M -1 1
1.0 0.8 1/m Lapp ( 10-9 mol -1 ) 0.6 0.4 0.2 0.0 1/m Lapp (x10-9 mol -1 ) 0 5 10 15 20 25 0.2 0.1 1/[Glipizide] ( 10-5 M -1 ) 0 0 2 4 1/[Glipizide] ( 10-5 M -1 ) Figure S1. Results of frontal analysis experiments for the binding of glipizide to a 2.0 cm 2.1 mm i.d. normal HSA column, as analyzed according to a double-reciprocal plot and Eq. (4). These results are for twelve glipizide concentrations that ranged from 0.5 to 50 µm. The inset shows the deviations occurring at low 1/[Glipizide] values from the best-fit line (as given in the above graph) that was determined from the linear region at high values of 1/[Glipizide]. 2
for glipizide at Sudlow site I of normal HSA. The binding of glipizide to Sudlow site I of ghsa1 and ghsa2 also gave linear relationships over a similar concentration range and when plotted according to Eq. (6). The correlation coefficients for these linear regions were 0.9854 (n = 5) and 0.9999 (n = 4), respectively. The association equilibrium constants that were determined for these regions were 4.4 (± 0.4) 10 4 M -1 for ghsa1 and 6.7 (± 0.1) 10 4 M -1 for ghsa2. The linear plots that were obtained by fitting Eq. (7) and that are described in the Table 2 and the main text for glipizide and R-warfarin were obtained at moderate-to-high concentrations of glipizide. When similar plots of k 0 /(k - k 0 ) vs. 1/[Glipizide] were made over the entire data set, linear relationships were still obtained with negative slopes and coefficients that ranged from 0.9933 to 0.9980 (n = 7). The association equilibrium constant and coupling constant that were obtained from the expanded fit for glipizide at Sudlow Site I on normal HSA was 8.9 (± 1.4) 10 4 M -1 and 0.32 (± 0.05). The corresponding values for ghsa1 were 20.0 (± 2.9) 10 4 M -1 and 0.62 (± 0.05), while the values for ghsa2 were 15.2 (± 2.0) 10 4 M -1 and 0.44 (± 0.05). Reverse Zonal Elution Competition Studies using Glipizide and Warfarin In the reverse zonal elution competition studies that were conducted between glipizide and warfarin, the part of the retention factor that was due to the interaction of glipizide at Sudlow Site II was estimated by using Eq. (9) along with the binding parameters given for glipizide at this site in Table 2 and the measured retention of L-tryptophan in the absence of glipizide. The contribution to the retention due to the weak affinity sites for glipizide was estimated by using Eq. (9) and the binding parameters in Table 1 that were measured for glipizide at these sites. The interaction at Sudlow Site II was found from these estimates to make up 11-40% of the total retention seen for glipizide on the normal HSA column in the presence of the various applied 3
concentrations of warfarin; the weak affinity interactions made up 2-6% of the total retention under the same conditions. Plots of the inverse of the corrected retention factor for glipizide, as obtained in these reverse competition studies, were made versus the concentration of warfarin in the mobile phase. A typical result is provided in Fig. S2, which provided a linear fit for the normal HSA column with a correlation coefficient of 0.9874 (n = 8). This linear fit confirmed that glipizide was competing with warfarin at Sudlow site I. Similar results were obtained for the ghsa1 and ghsa2 columns, which gave correlation coefficients of 0.9917-0.9930 (n = 8). The association equilibrium constant that was estimated for warfarin from these plot were in the range of 1.2-1.3 10 5 M -1, which was comparable to a previously-reported association equilibrium constant for warfarin at Sudlow site I of 2.4 (± 0.4) 10 5 M -1 [S1,S2]. The association equilibrium constants that were measured for glipizide at Sudlow site I from the same plots were 8.4-9.5 10 5 M -1, which also should reasonable agreement with the results that had been obtained by normal zonal elution competition studies. Reverse Zonal Elution Competition Studies using Glipizide and Tamoxifen Reverse competition studies similar to those described in the previous section were also conducted in which tamoxifen was used as the competing agent and glipizide was the injected probe. In this case, the retention factor due to interactions at Sudlow site II made up 11-42% of the total retention seen for glipizide, while the weak affinity interactions made up 2-6%. Plots of the inverse of the corrected retention factor for glipizide versus the concentration of tamoxifen in the mobile phase resulted in non-linear plots (see Fig. S3), as observed for both normal HSA and glycated HSA. This behavior again suggested that allosteric interactions were present between glipizide and tamoxifen during their binding to normal HSA and glycated HSA. 4
0.08 1/k(Glipizde,Corrected) 0.06 0.04 0.02 0.00 0 5 10 15 20 [Warfarin] ( 10-6 M) Figure S2. A typical plots of 1/(k Glipizide, Corrected ) versus concentration of warfarin, as obtained in reverse competition studies. These results are for eight warfarin concentrations that ranged from 0 to 20 µm.. The equation for the best-fit line was y = [2.6 (± 0.2) 10 3 ] x + [0.02 (± 0.01)], with a correlation coefficient of 0.9874 (n = 8). The error bars represent a range of ± 1 S.D. Each point is the average of four values with relative standard deviations that ranged from ± 0.3-6.1%. 5
0.08 1/k(Glipizide,Corrected) 0.06 0.04 0.02 0.00 0 5 10 [Tamoxifen] ( 10-6 M) Figure S3. Results of plots of 1/(k Glipizide,Corrected ) versus the concentration of tamoxifen. These results are for eight warfarin concentrations that ranged from 0 to 10 µm. The error bars represent a range of ± 1 S.D. Each point is the average of four values with relative standard deviations that ranged from ± 0.7-3.2%. 6
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