Development Of An Affinity Silica Monolith Containing α1- Acid Glycoprotein For Chiral Separations

An affinity monolith based on silica and containing immobilized α1-acid glycoprotein (AGP) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of AGP in the silica monolith was 18% higher than that obtained with silica particles and 61% higher than that measured for a GMA/EDMA monolith. The higher surface area of the silica monolith gave materials that contained 1.5- to 3.6- times more immobilized protein per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the AGP silica monolith were evaluated by injecting two chiral analytes onto this column (i.e., R/S-warfarin and R/S-propranolol). In each case, the AGP silica monolith gave higher retention plus better resolution and efficiency than AGP columns containing silica particles or a GMA/EDMA monolith. The AGP silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with AGP as a chiral stationary phase

Studies of Verapamil Binding to Human Serum Albumin By High-Performance Affinity Chromatography

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body

Studies of Verapamil Binding to Human Serum Albumin By High-Performance Affinity Chromatography

The binding of verapamil to the protein human serum albumin (HSA) was examined by using high performance affinity chromatography. Many previous reports have investigated the binding of verapamil with HSA, but the exact strength and nature of this interaction (e.g., the number and location of binding sites) is still unclear. In this study, frontal analysis indicated that at least one major binding site was present for R- and S-verapamil on HSA, with estimated association equilibrium constants on the order of 104 M−1 and a 1.4-fold difference in these values for the verapamil enantiomers at pH 7.4 and 37°C. The presence of a second, weaker group of binding sites on HSA was also suggested by these results. Competitive binding studies using zonal elution were carried out between verapamil and various probe compounds that have known interactions with several major and minor sites on HSA. R/S-Verapamil was found to have direct competition with S-warfarin, indicating that verapamil was binding to Sudlow site I (i.e., the warfarin-azapropazone site of HSA). The average association equilibrium constant for R- and S-verapamil at this site was 1.4 (±0.1) × 104 M−1. Verapamil did not have any notable binding to Sudlow site II of HSA but did appear to have some weak allosteric interactions with L-tryptophan, a probe for this site. An allosteric interaction between verapamil and tamoxifen (a probe for the tamoxifen site) was also noted, which was consistent with the binding of verapamil at Sudlow site I. No interaction was seen between verapamil and digitoxin, a probe for the digitoxin site of HSA. These results gave good agreement with previous observations made in the literature and help provide a more detailed description of how verapamil is transported in blood and of how it may interact with other drugs in the body

Experimental limits on monojet production in e+e- annihilation at 29 GeV

A search was made for e+e--->X1X2 where X1 consists of one or more light unobservable particles and X2 decays promptly to a visible jet of particles. One event was found for an integrated luminosity of 176 pb-1, a rate consistent with known backgrounds. This result places a significant constraint on a number of theoretical models.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/25641/3/0000191.pd

Development of monolithic supports and improved immobilization methods for high -performance affinity chromatography and free drug analysis

This work combines five projects. In the first project, affinity monoliths based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA) were developed for ultrafast immunoextractions. Rabbit immunoglobulin G (IgG) and anti-FITC antibodies were used as model ligands for this work. The antibody content of the monoliths was optimized by varying both the polymerization and immobilization conditions for preparing such supports. When a 4.5 mm i.d. × 0.95 mm monolith disk containing anti-FITC antibodies was used, 95% extraction of fluorescein was achieved in 100 ms. In the second project, several immobilization methods were explored for the preparation of high-performance affinity monolithic columns containing human serum albumin (HSA). These monoliths were based on a copolymer of GMA and EDMA. Each HSA monolith was evaluated in terms of its total protein content and its retention of (R/S)-warfarin and D/L-tryptophan. In the third project, affinity monoliths based on silica and containing immobilized HSA or α1-acid glycoprotein (AGP) were developed and evaluated in terms of their binding, efficiency and selectivity in chiral separations. It was found that the amount of immobilized protein per unit volume, retention, efficiency and resolving power (for chiral drugs) of silica monoliths containing HSA or AGP were better than columns containing silica particles or GMA/EDMA monoliths. The fourth project introduced two techniques using maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method) for the immobilization of HSA and other ligands to silica through sulfhydryl groups. A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. The fifth project introduced a new ultrafast extraction technique for free drug analysis and determination of association equilibrium constants for drug-protein binding. Some advantages of this method include its speed (approximately 5 min per run) and its ability to use the same affinity column for more than one type of drug

Development Of Sulfhydryl-Reactive Silica For Protein Immobilization In High-Performance Affinity Chromatography

Two techniques were developed for the immobilization of proteins and other ligands to silica through sulfhydryl groups. These methods made use of maleimide-activated silica (the SMCC method) or iodoacetyl-activated silica (the SIA method). The resulting supports were tested for use in highperformance affinity chromatography by employing human serum albumin (HSA) as a model protein. Studies with normal and iodoacetamide-modified HSA indicated that these methods had a high selectivity for sulfhydryl groups on this protein, which accounted for the coupling of 77–81% of this protein to maleimide- or iodacetyl-activated silica. These supports were also evaluated in terms of their total protein content, binding capacity, specific activity, non-specific binding, stability and chiral selectivity for several test solutes. HSA columns prepared using maleimide-activated silica gave the best overall results for these properties when compared to HSA that had been immobilized to silica through the Schiff base method (i.e., an amine-based coupling technique). A key advantage of the supports developed in this work is that they offer the potential of giving greater site-selective immobilization and ligand activity than amine-based coupling methods. These features make these supports attractive in the development of protein columns for such applications as the study of biological interactions and chiral separations

Chromatographic analysis of carbamazepine binding to human serum albumin: II. Comparison of the Schiff base and \u3ci\u3eN\u3c/i\u3e-hydroxysuccinimide immobilization methods

Recent studies with carbamazepine on human serum albumin (HSA) columns have noted an appreciable degree of non-specific binding on supports prepared by the Schiff base immobilization method. This work examines an alternative immobilization method for HSA based on N-hydroxysuccinimide (NHS)-activated silica. This support was prepared by reacting HPLC-grade silica directly with disuccinimidyl carbonate. The resulting material was compared to an HSA support prepared by the Schiff base method in terms of its activity for carbamazepine and non-specific interactions with this drug. When examined by frontal analysis, both supports gave comparable association equilibrium constants for carbamazepine interactions with HSA ((0.53–0.55) × 104 M−1 at 37 °C). However, columns prepared by the Schiff base method gave greater non-specific binding. These columns, as well as control columns prepared using the carbonyldiimidazole (CDI) immobilization method, were also evaluated for their non-specific binding to a variety of other solutes known to interact with HSA. From these results it was concluded that the NHS method was an attractive alternative to the Schiff base technique in the preparation of immobilized HSA for HPLC-based binding studies for carbamazepine. However, it was also noted that nonspecific binding varies from one drug to the next in these immobilization methods, indicating that such properties should be evaluated on a case-by-case basis in the use and development of HSA columns for binding studies

Immobilization Of Alpha 1-Acid Glycoprotein For Chromatographic Studies Of Drug-Protein Binding Ii. Correction For Errors In Association Constant Measurements

A new method for the immobilization of α1-acid glycoprotein (AGP) in HPLC columns was recently described for applications such as drug binding studies. Part of this earlier work used self-competition zonal elution studies to measure association equilibrium constants between immobilized AGP and R- or S-propranolol. It was later found that analysis of these data by a common equation derived for linear elution conditions gave erroneous values for experiments actually conducted under nonlinear conditions. This report discusses the nature of this error and uses frontal analysis to estimate the true binding strength between R- and S-propranolol and HPLC columns containing immobilized AGP

Development of an Affinity Silica Monolith Containing Human Serum Albumin For Chiral Separations

An affinity monolith based on silica and containing immobilized human serum albumin (HSA) was developed and evaluated in terms of its binding, efficiency and selectivity in chiral separations. The results were compared with data obtained for the same protein when used as a chiral stationary phase with HPLC-grade silica particles or a monolith based on a copolymer of glycidyl methacrylate (GMA) and ethylene dimethacrylate (EDMA). The surface coverage of HSA in the silica monolith was similar to values obtained with silica particles and a GMA/EDMA monolith. However, the higher surface area of the silica monolith gave a material that contained 1.3- to 2.2-times more immobilized HSA per unit volume when compared to silica particles or a GMA/EDMA monolith. The retention, efficiency and resolving power of the HSA silica monolith were evaluated using two chiral analytes: D/L-tryptophan and R/S-warfarin. The separation of R- and S-ibuprofen was also considered. The HSA silica monolith gave higher retention and higher or comparable resolution and efficiency when compared with HSA columns that contained silica particles or a GMA/EDMA monolith. The silica monolith also gave lower back pressures and separation impedances than these other materials. It was concluded that silica monoliths can be valuable alternatives to silica particles or GMA/EDMA monoliths when used with immobilized HSA as a chiral stationary phase