Revealing Equilibrium and Rate Constants of Weak and Fast Noncovalent Interactions

Rate and equilibrium constants of weak noncovalent molecular interactions are extremely difficult to measure. Here, we introduced a homogeneous approach called equilibrium capillary electrophoresis of equilibrium mixtures (ECEEM) to determine kon, koff, and Kd of weak (Kd > 1 μM) and fast kinetics (relaxation time, τ 1000 filled with T. Afterward, differential mobility of L, T, and C along the reactor is induced by an electric field. The combination of differential mobility of reactants and their interactions leads to a change of the EM peak shape. This change is a function of rate constants, so the rate and equilibrium constants can be directly determined from the analysis of the EM peak shape (width and symmetry) and propagation pattern along the reactor. We proved experimentally the use of ECEEM for multiplex determination of kinetic parameters describing weak (3 mM > Kd > 80 μM) and fast (0.25 s ≥ τ ≥ 0.9 ms) noncovalent interactions between four small molecule drugs (ibuprofen, S-flurbiprofen, salicylic acid and phenylbutazone) and α- and β-cyclodextrins. The affinity of the drugs was significantly higher for β-cyclodextrin than α-cyclodextrin and mostly determined by the rate constant of complex formation

Viral Quantitative Capillary Electrophoresis for Counting Intact Viruses

The quantification of a virus plays an important role in vaccine development, clinical diagnostics, and environmental contamination assays. In all these cases, it is essential to calculate the concentration or number of intact virus particles (ivp) and estimate the degree of degradation and contamination of virus samples. In this work, we propose a cost-efficient, robust method for the quantification and characterization of intact viruses based on capillary zone electrophoresis. This separation method is demonstrated on vaccinia virus (VV) with oncolytic properties. After virus sample preparation, the solution contains intact VV as well as broken viruses and residual DNA from the host cell used for preparation. Regulatory requirements limit the amount of the host cell DNA that can be present in vaccines or human therapeutics. We apply capillary electrophoresis to separate intact virus particles and the residual DNA and to measure the level of virus contamination with DNA impurities. Intercalating YOYO-1 dye is used to detect the encapsulated and free DNA by laser-induced fluorescence. After soft lysis of VV with proteinase K, all encapsulated DNA is dissolved to the free DNA. The change in peak areas and a DNA calibration curve help determine the initial concentration of intact viruses. This viral quantitative capillary electrophoresis (Viral qCE) is able to quantify the oncolytic vaccinia virus in the range of 106 to 1012 ivp/mL

Viral Quantitative Capillary Electrophoresis for Counting and Quality Control of RNA Viruses

The world of health care has witnessed an explosive boost to its capacity within the past few decades due to the introduction of viral therapeutics to its medicinal arsenal. As a result, a need for new methods of viral quantification has arisen to accommodate this rapid advancement in virology and associated requirements for efficiency, speed, and quality control. In this work, we apply viral quantitative capillary electrophoresis (viral qCE) to determine (i) the number of intact virus particles (ivp) in viral samples, (ii) the amount of DNA contamination, and (iii) the degree of viral degradation after sonication, vortexing, and freeze–thaw cycles. This quantification method is demonstrated on an RNA-based vesicular stomatitis virus (VSV) with oncolytic properties. A virus sample contains intact VSV particles as well as residual DNA from host cells, which is regulated by WHO guidelines, and may include some carried-over RNA. We use capillary zone electrophoresis with laser-induced fluorescent detection to separate intact virus particles from DNA and RNA impurities. YOYO-1 dye is used to stain all DNA and RNA in the sample. After soft lysis of VSV with proteinase K digestion of viral capsid and ribonucleoproteins, viral RNA is released. Therefore, the initial concentration of intact virus is calculated based on the gain of a nucleic acid peak and an RNA calibration curve. After additional NaOH treatment of the virus sample, RNA is hydrolyzed leaving residual DNA only, which is also calculated by a DNA calibration curve made by the same CE instrument. Viral qCE works in a wide dynamic range of virus concentrations from 10⁸ to 10¹³ ivp/mL. It can be completed in a few hours and requires minimum optimization of CE separation

Bioanalysis for Biocatalysis: Multiplexed Capillary Electrophoresis–Mass Spectrometry Assay for Aminotransferase Substrate Discovery and Specificity Profiling

In this work, we introduce an entirely automated enzyme assay based on capillary electrophoresis coupled to electrospray ionization mass spectrometry termed MINISEP-MS for multiple interfluent nanoinjections–incubation–separation–enzyme profiling using mass spectrometry. MINISEP-MS requires only nanoliters of reagent solutions and uses the separation capillary as a microreactor, allowing multiple substrates to be assayed simultaneously. The method can be used to rapidly profile the substrate specificity of any enzyme and to measure steady-state kinetics in an automated fashion. We used the MINISEP-MS assay to profile the substrate specificity of three aminotransferases (<i>E. coli</i> aspartate aminotransferase, <i>E. coli</i> branched-chain amino acid aminotransferase, and <i>Bacillus sp.</i> YM-1 d-amino acid aminotransferase) for 33 potential amino acid substrates and to measure steady-state kinetics. Using MINISEP-MS, we were able to recapitulate the known substrate specificities and to discover new amino acid substrates for these industrially relevant enzymes. Additionally, we were able to measure the apparent <i>K</i>_M and <i>k</i>_cat parameters for amino acid donor substrates of these aminotransferases. Because of its many advantages, the MINISEP-MS assay has the potential of becoming a useful tool for researchers aiming to identify or create novel enzymes for specific biocatalytic applications