Dataset Information

Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy.

ABSTRACT: Water from the solvent very strongly absorbs light in the frequency range of interest for studying protein structure by infrared (IR) spectroscopy. This renders handling of the observation cells painstaking and time consuming, and limits the reproducibility of the measurements when IR spectroscopy is applied to proteins in aqueous solutions. These difficulties are circumvented by the use of an Attenuated Total Reflectance (ATR) accessory. However, when protein solutions are studied, ATR spectroscopy suffers from several drawbacks, the most severe being nonproportionality of the observed absorbance with the protein concentration and spectral distortions that vary from protein to protein and from sample to sample. In this study, we show (1) that the nonproportionality is due to adsorption of the protein on the ATR crystal surface; (2) that the contribution of the crystal-adsorbed protein can easily be taken into account, rendering the corrected absorbance proportional to the protein concentration; (3) that the observed variable base line distortions, likely due to changes in the penetration depth of the light beam in solutions with the refractive index that depends on the protein concentration, can be easily eliminated; and (4) that ATR IR spectra thus corrected for protein adsorption and light penetration can be used to properly analyze the secondary structure of proteins in solution.

SUBMITTER: Goldberg ME

PROVIDER: S-EPMC2253228 | biostudies-literature | 2005 Nov

REPOSITORIES: biostudies-literature

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Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy.

Goldberg Michel E ME Chaffotte Alain F AF

Protein science : a publication of the Protein Society 20051101 11

Water from the solvent very strongly absorbs light in the frequency range of interest for studying protein structure by infrared (IR) spectroscopy. This renders handling of the observation cells painstaking and time consuming, and limits the reproducibility of the measurements when IR spectroscopy is applied to proteins in aqueous solutions. These difficulties are circumvented by the use of an Attenuated Total Reflectance (ATR) accessory. However, when protein solutions are studied, ATR spectros ...[more]

PMID: 16251363

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Project description:BACKGROUND:The topical HIV prevention (microbicides) field is in acute need of a method to rapidly and objectively measure adherence to product use in clinical trials. Infrared (IR) spectroscopy has been used in many pharmaceutical and forensic applications but has yet to be applied to adherence monitoring. In this study, we report on efforts to test the feasibility of using IR spectroscopy as a means to measure residual active or placebo vaginal product, semen exposure and vaginal insertion from a single swab. METHODS:A portable IR spectrometer equipped with diamond attenuated total reflectance (ATR) was used to capture spectra of unused vs. vaginally-used swabs, vaginal swabs containing semen, and vaginal swabs to which either tenofovir-containing or matching placebo products (vaginal gel or insert) were added. Spectral data obtained from swabs placed directly on the spectrometer were divided into calibration and testing sets for developing and validating discriminant models set up to provide yes/no predictions of: vaginal vs. non-vaginal use, presence vs. no presence of each test product, and presence vs. no presence of semen. Further validation of models was performed using vaginal swabs collected from a clinical study evaluating vaginally administered placebo insert formulations. RESULTS:For each discriminant model developed to predict vaginal vs. non-vaginal use, presence vs. no presence of each test product, and presence vs. no presence of semen, classified validation samples not included in the model development were correctly identified into their respective classes with minimal prediction error. Clinically obtained vaginal swabs collected 15-60 minutes after placebo insert use were also correctly identified, further validating the models. CONCLUSION:Our findings demonstrate the proof of concept that IR spectroscopy can be a method for rapid detection and characterization of microbicide products and biological fluids present in vaginal swabs. This novel method has potential to support real-time, on-site adherence monitoring in clinical or field settings.

Dataset Information

Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy.

Publications

Undistorted structural analysis of soluble proteins by attenuated total reflectance infrared spectroscopy.

Similar Datasets

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