The Modification of Carboxyl Groups
Roger L. Lundblad, Claudia M. Noyes in Chemical Reagents for Protein Modification, 1984
There are several observations on the use of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Figure 9 compares the rate of inactivation of yeast enolase35 with this reagent and several other carbodimides. An exogenous nucleophile was not used in these experiments. Note that 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC) appears to be far more effective than the other two carbodiimides under these reaction conditions. The selective modification of a single aspartyl residue at the active site of lysozyme (Asp-101) was accomplished by the use of a low molar excess (five to tenfold) of carbodiimide. A variety of attacking nucleophiles were used in this study (the modification reactions were performed at pH 5.0 maintained with HCl during the reaction). Of particular interest is the success achieved in the separation of the products of the reaction by ion-exchange chromatography as shown in Figure 10 and Figure 11. The modification of pancreatic phospholipase A2 (Figure 12) provides a particularly useful example of the effect of pH on carbodiimide-mediated modification. Note that the inactivation is much more rapid at pH 3.5 than at pH 5.5.
The Modification of Carboxyl Groups
Roger L. Lundblad, Claudia M. Noyes in Chemical Reagents for Protein Modification, 1984
There are several observations on the use of l-ethyl-3-(3-dimefhylaminopropyl)-carbo-diimide (EDC). Figure 9 compares the rate of inactivation of yeast enolase35 with this reagent and several other carbodimides. An exogenous nucleophile was not used in these experiments. Note that l-ethyl-3-(4-azonia4,4-dimethylpentyl)-carbodiimide iodide (EAC) appears to be far more effective than the other two carbodiimides under these reaction conditions. The selective modification of a single aspartyl residue at the active site of lysozyme (Asp-101) was accomplished by the use of a low molar excess (five to tenfold) of carbodiimide. A variety of attacking nucleophiles were used in this study (the modification reactions were performed at pH 5.0 maintained with HCl during the reaction). Of particular interest is the success achieved in the separation of the products of the reaction by ion-exchange chromatography as shown in Figure 10 and Figure 11. The modification of pancreatic phospholipase A2 (Figure 12) provides a particularly useful example of the effect of pH on carbodiimide -mediated modification. Note that the inactivation is much more rapid at pH 3.5 than at pH 5.5.
The Modification Of Carboxyl Groups
Roger L. Lundblad in Chemical Reagents for Protein Modification, 2020
There are several observations on the use of l-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC). Figure 14 compares the rate of inactivation of yeast enolase28 with this reagent and several other carbodimides. An exogenous nucleophile was not used in these experiments. Note that l-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide (EAC) appears to be far more effective than the other two carbodiimides under these reaction conditions. In the absence of added nucleophile, the carbodiimide-activated protein carboxyl group may rearrange (Figure 15) to form a substituted O-acylurea as described by Borders and co-workers for the modification of thrombin by various water-soluble carbodiimides.29,30 The selective modification of a single aspartyl residue at the active site of lysozyme (Asp-101)31 was accomplished by the use of a low molar excess (five- to tenfold) of carbodiimide. A variety of attacking nucleophiles were used in this study (the modification reactions were performed at pH 5.0 maintained with HCl during the reaction). Of particular interest is the success achieved in the separation of the products of the reaction by ion-exchange chromatography as shown in Figure 16 and Figure 17. The modification of pancreatic phospholipase A2 (Figure 18) provides a particularly useful example of the effect of pH on carbodiimide-mediated modification. Note that the inactivation is much more rapid at pH 3.5 than at pH 5.5.
Nanoparticle-protein corona complex: understanding multiple interactions between environmental factors, corona formation, and biological activity
Published in Nanotoxicology, 2021
Aysel Tomak, Selin Cesmeli, Bercem D. Hanoglu, David Winkler, Ceyda Oksel Karakus
Differential centrifugal sedimentation (DCS) is a less commonly used particle sizing method with relatively high sensitivity. Krpetic et al. (2013) used DCS to measure the particle sizes of ligand stabilized AuNPs in the 10–50 nm range, noting its high sensitivity in detecting extremely small shell thickness variations in NPs. Similar high accuracy was achieved by Röcker et al. (2009) who successfully employed an alternative technique, fluorescence correlation spectroscopy (FCS), to quantify the adsorption of human serum albumin onto very small (<20 nm) polymer-coated NPs. Another analytical procedure traditionally used to provide information on protein fragments and aggregates is size-exclusion chromatography (SEC). This separates proteins and other molecules based on their size using their retention times. It has been successfully applied to numerous tasks, such as characterizing protein biopharmaceuticals and industrial polymers (Hong, Koza, and Bouvier 2012). It can potentially be used to determine the presence and thickness of protein corona on NPs. However, it has not been validated for use in studying structural properties of NP-protein corona complexes, due to decreased sensitivity at low concentrations of high molecular weight NP aggregates. Ion-exchange chromatography (IEC) is also frequently used for separating biomolecules of different charges. However, only a few studies involving NP-protein interactions have been reported, mainly for purifying NPs based on charge (Lévy et al. 2004).
Liquid chromatography coupled to mass spectrometry for metabolite profiling in the field of drug discovery
Published in Expert Opinion on Drug Discovery, 2019
Javier Saurina, Sonia Sentellas
Ion exchange chromatography results in a good choice for the separation of permanent or pH-dependent charged analytes. A scheme of some popular stationary phases can be seen in Figure 2(b). Strong sulfonic and quaternary ammonium columns are ionized at any pH of the mobile phase thus interacting, respectively, with cationic and anionic drugs and metabolites. Weak exchangers correspond to carboxylic and amino groups which are protonated or deprotonated depending on the pH of the mobile phase. A recent example of anion exchange concerned the study of highly polar metabolites of BMS-986094 in plasma and tissues using LC with tandem mass spectrometry (MS/MS) detection [40]. Nucleoside triphosphates negatively charged in physiological media were separated by mixed-mode combining C18 and weak ion exchange groups. Another paper described the characterization of thiopurine metabolites in blood samples by anion exchange LC-MS/MS [41]. Thiopurine phosphorylated species were separated on a polyethyleneimine exchanger using an elution program based on increasing pH from 6.0 to 10.5.
New generation of viral nanoparticles for targeted drug delivery in cancer therapy
Published in Journal of Drug Targeting, 2022
Nikta Alvandi, Maryam Rajabnejad, Zeynab Taghvaei, Neda Esfandiari
After VLPs assembly process, VLPs should be released from the host cells by different mechanisms which depend on VLPs structure. As shown by Figure 3(B), generally for obtaining VLPs from the culture medium or i.e. purification, three steps were recorded as follows: cell lysis and clarification, intermediate purification, and polishing. The first step initiates using 1% triton X-100 after harvesting of cell culture. Triton X-100 is a detergent utilised for cell lysis and protein extraction. Then, removing cell debris and aggregates is designed as a clarification process by centrifugation. Afterward, the intermediate purification step begins that concentration has a critical role in this process. Accordingly, adsorptive chromatography is a great choice for this step. Moreover, other chromatographic matrices based on porous membrane layers are utilised too. It should be noted that these porous matrices are devoted to large particles like VLPs. In the last step, polishing, residual host-cell protein, and DNA are removed. In this step, affinity and ion-exchange chromatography are suited. Sometimes size-exclusion chromatography is used too like when VLPs-derived impurities, such as non-assembled proteins have similar electrostatic features with VLPs. After obtaining purified VLPs, ultracentrifugation is occurred in some studies [36].
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