Molecular Characterization of Recombinant Human Inhibin α- and βB-Subunits and Activin A (βA-βA) Using Raman Spectroscopy

Members of the transforming growth factor-β (TGF-β) superfamily, including inhibins and activins, are structurally related signaling proteins that regulate reproductive and endocrine physiology. Although these proteins share a conserved cystine-knot architecture, differences in subunit composition and molecular organization contribute to distinct biological activities and can complicate their analytical discrimination. Here, Raman spectroscopy was used to characterize recombinant human inhibin α-subunit (INHA), recombinant human inhibin βB-subunit (INHBB (βB-subunit)), and activin A (βA-βA homodimer) under identical experimental conditions using 532 nm excitation. Raman spectra were baseline-corrected, Savitzky–Golay-smoothed, and area-normalized. Distinct spectral features were observed across the 500–1800 cm−1 protein fingerprint region, particularly in the S–S stretching, aromatic amino acid residue, amide III, and amide I regions. The S–S stretching region showed the highest relative contribution in INHA, whereas differences in phenylalanine- and amide-associated bands further distinguished the three proteins. These findings show that Raman spectra may contain molecular information that can distinguish closely related inhibin/activin proteins under the investigated solid-state conditions. Raman spectroscopy therefore provides a label-free, complementary approach to the molecular fingerprinting of TGF-β superfamily proteins and may facilitate their molecular characterization.