Raman optical activity characterization of native and molten globule states of equine lysozyme: Comparison with hen lysozyme and bovine α-lactalbumin

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Abstract
Vibrational Raman optical activity (ROA) spectra of the calcium‐binding lysozyme from equine milk in native and nonnative states are measured and compared with those of the homologous proteins hen egg white lysozyme and bovine α‐lactalbumin. The ROA spectrum of holo equine lysozyme at pH 4.6 and 22°C closely resembles that of hen lysozyme in regions sensitive to backbone and side chain conformations, indicating similarity of the overall secondary and tertiary structures. However, the intensity of a strong positive ROA band at ∼1340 cm−1, which is assigned to a hydrated form of α helix, is more similar to that in the ROA spectrum of bovine α‐lactalbumin than hen lysozyme and may be associated with the greater flexibility and calcium‐binding ability of equine lysozyme and bovine α‐lactalbumin compared with hen lysozyme. In place of a strong sharp positive ROA band at ∼1300 cm−1 in hen lysozyme that is assigned to an α helix in a more hydrophobic environment, equine lysozyme shows a broader band centered at ∼1305 cm−1, which may reflect greater heterogeneity in some α‐helical sequences. The ROA spectrum of apo equine lysozyme at pH 4.6 and 22°C is almost identical to that of the holo protein, which indicates that loss of calcium has little influence on the backbone and side chain conformations, including the calcium‐binding loop. From the similarity of their ROA spectra, the A state at pH 1.9 and both 2 and 22°C and the apo form at pH 4.5 and 48°C, which are partially folded denatured (molten globule or state A) forms of equine lysozyme, have similar structures that the ROA suggests contain much hydrated α helix. The A state of equine lysozyme is shown by these results to be more highly ordered than that of bovine α‐lactalbumin, the ROA spectrum of which has more features characteristic of disordered states. A positive tryptophan ROA band at ∼1551 cm−1 in the native holo protein disappears in the A state, which is probably due to the presence of nonnative conformations of the tryptophans associated with a previously identified cluster of hydrophobic residues.