Abstract
The Raman spectra of β‐lactoglobulin in the crystalline, freeze‐dried, and solution states are compared. The spectra of the freeze‐dried and crystalline proteins were practically identical. The conformationally sensitive amide III line appearing at 1242 cm^−1^ increased in intensity 30% upon dissolution of the protein in water which is interpreted as a conformational change in the disordered chains of the protein. This result appears to be a phenomenon for globular proteins containing a large disordered chain fraction.
The alkaline denaturation of β‐lactoglobulin was studied. When the pH was increased from 6.0 to 11.0, the amide III line shifted from 1242 to 1246 cm^−1^, broadened, and decreased in intensity. This is consistent with the conversion of β‐sheet regions in β‐lactoglobulin to the disordered conformation, as has been proposed by other investigators. At pH 13.5 the amide III shifts to 1257 cm^−1^ characteristic of a completely disordered protein, indicating that any remaining “core” of β‐sheet has been randomized.
Several changes in the intensities of the tyrosine and tryptophan vibrations accompany the denaturation. As the pH is increased from 6.0 (native state) to 11.0 (denatured state) the intensity ratio of two tyrosine ring vibrations, I~855~ cm^−1^/I~830~ cm^−1^, decreases from 1.0:0.9 to 1.0:1.3. The same ratio for a copolymer consisting of 95% glutamic acid and 5% tyrosine at pH 7.0, where the polymer forms a random coil exposing the tyrosine to the aqueous environment, is 1.0:0.62. This ratio more closely resembles that corresponding to β‐lactoglobulin at pH 6.0 (native state) than pH 11.0 (denatured state) suggesting that the average tyrosine in the denatured state may be in a more hydrophobic environment than in the native state. A time‐dependent polymerization of the denatured protein reported by other investigators and observed by us may account for the change in the tyrosine environment.
A tryptophan vibration appearing at 833 cm^−1^ in the spectrum of the native state becomes weak as the pH is increased to 11.0. The intensity of this line may also reflect the local environment of the tryptophan residue.