Matthew B. Kubilius
2018
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Abstract
1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide (EDC) is a commonly used reagent for bioconjugation and peptide synthesis. Both EDC and the corresponding urea derivative, 1-(3-dimethylaminopropyl)-3-ethylurea (EDU), are achiral. As the reagent is active in aqueous solutions, it is a common choice for the study of evolving secondary structural changes via circular dichroism. This work highlights the effect of EDU on spectropolarimetric measurements, namely, the problematic absorption profile at low wavelengths (190−220 nm). We demonstrate that EDU is capable of erroneously indicating structural changes, particularly loss of α-helical character, through masking of the characteristic minimum at 208 nm. However, if the concentrations of the EDU in the sample are known, then this effect can be anticipated and calculations of secondary structure can be adjusted to avoid the impacted wavelengths. Impacts of EDU in a sample are compared to those of standard urea, which, by contrast, is commonly used as a denaturant in circular dichroism studies without issue. ■ INTRODUCTION This work highlights the influence of 1-(3-dimethylaminopropyl)-3-ethylurea (EDU) in circular dichroism (CD) measurements. The importance of this work stems from the ubiquity of the ca rbod i im ide coup l ing agen t , 1 e thy l 3 [3 dimethylaminopropyl]carbodiimide (EDC) in bioconjugation. The presence of EDU in CD samples can cause the underlying data to be obscured, preventing the interpretation of structure for protein and peptide bioconjugates. A key advantage of EDC bioconjugation is its compatibility with aqueous solutions, making its conjugation chemistry an attractive choice for study of evolving secondary structural changes using circular dichroism. For example, recent work has demonstrated a desire to study the self-assemblies of small (three amino acid) peptide sequences computationally and experimentally.With an understanding of the influence of the linking reagents on these measurements, these self-assemblies have the potential to be measured as they are grown. EDC is used as a linking reagent for condensation reactions linking amines to carboxylic acid groups, typically in aqueous media. The EDU product of this reaction is the urea form of the original carbodiimide molecule, as shown in Figure 1. Because of the molecule’s ability to react with both water and the molecule of interest, EDC usage protocols generally recommend a 10:1 molar ratio of EDC to each carboxylic acid, yielding these same elevated concentrations of EDU in solution post-reaction. Both EDC and EDU are achiral, and they might be assumed not to directly impact CD measurement. Unlike urea, however, EDU’s applicable concentration range, absorption impact, and wavelength range of interference on circular dichroism measurements are not commonly known. Establishing those parameters will allow for the development of well-controlled studies with clearly understood results using this reagent in solution. Typically, the issues explored in this work are eliminated in advance of structural studies by chromatography purification of samples post-conjugation. Although that method is undeniably effective, it is incompatible with kinetic studies or attempts to directly measure the change in the secondary structure as a function of reaction completion in real time. We characterize the effect of EDU in a solution by direct inspection of CD signals of the common reference samples bovine serum albumin (BSA) and pantolactone measured in increasing concentrations of both urea and EDU. By evaluating the two-component system, we quantify the degree that standard urea can be used without issue, whereas the EDU can be problematic over similar concentration ranges. Because circular dichroism measures the difference in absorbance of leftand right-polarized light, any components in a solution able to change this ratio will give rise to changes in the spectra obtained. In this work, we highlight the degree that EDU absorbs over a much broader range of wavelengths as well as the impact that this adsorption has on secondary structure interpretation. Because only chiral molecules or molecules with certain fixed orientations give rise to such a difference, small, achiral molecules do not generally impact circular dichroism measurements. However, if an achiral species is present in a sample that significantly absorbs Received: September 1, 2017 Accepted: October 13, 2017 Published: November 21, 2017 Article http://pubs.acs.org/journal/acsodf © 2017 American Chemical Society 8308 DOI: 10.1021/acsomega.7b01288 ACS Omega 2017, 2, 8308−8312 Cite This: ACS Omega 2017, 2, 8308-8312 This is an open access article published under an ACS AuthorChoice License, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.