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

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. (C) 2000 John Wiley and Sons, Inc.