In this study, MMS was used to accurately determine the higher-order structure of hemoglobin, a highly colored protein in solution, by interrogating IR spectra in the Amide I band region. The hemoglobin molecule is primarily composed of alpha-helices, with some turn and unordered structures and very few beta-sheets. Protein spectra were automatically acquired using a 24-well plate format and processed using the AQS³delta software and Data Analysis package.
Results were obtained from a range of hemoglobin concentrations with varying amounts of pigmentation demonstrating that the strong color intensity of the samples did not affect or interfere with spectral quality or the resulting higher order analysis of the protein. In addition, the secondary structure composition as determined by MMS was highly consistent with the results obtained from X-ray crystallography, the most widely used technique for resolving the 3-dimensional protein structure, and AlphaFold, the most accurate AI system for protein structural prediction.