Ultraviolet resonance Raman examination of horse apomyoglobin acid unfolding intermediates.
We have used UV resonance Raman spectroscopy to study the acid-induced denaturation of horse apomyoglobin (apoMb) between pH 7. 0 and 1.8. The 206.5 nm excited Raman spectra are dominated by amide vibrations, which are used to quantitatively determine the apoMb secondary structure. The 229 nm excited Raman spectra are dominated by the Tyr and Trp Raman bands, which are analyzed to examine changes of Tyr and Trp environments and solvent exposures. We observe two partially unfolded apoMb intermediates at pH 4 and pH 2, while we observe only one partially unfolded holoMb intermediate at 2, in which the G and H helices are mainly intact, while the rest of protein is unfolded. This partially unfolded holoMb intermediate at pH 2 is essentially identical to the pH 2 apoMb intermediate. The partially unfolded pH 4 apoMb intermediate is composed of the three folded A, G, and H helices and contains 38% helical structure. The changes in the Trp Raman cross sections during the acid-induced denaturation indicates that Trp 7 is likely to be fully exposed in the apoMb pH 4 intermediate and that the A helix melts with a pKa approximately 3.5.
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Department of Chemistry, Chevron Science Center, University of Pittsburgh, Pennsylvania 15260, USA.
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D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000378:metabolism*; D006736:Horses; D006851:Hydrochloric Acid; D006863:Hydrogen-Ion Concentration; D008786:Metmyoglobin / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry* / Q000378:metabolism*; D011489:Protein Denaturation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D013053:Spectrophotometry; D013059:Spectrum Analysis, Raman / Q000379:methods; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry
D001059:Apoproteins; D009211:Myoglobin; C010857:apomyoglobin; D008786:Metmyoglobin; D014443:Tyrosine; D014364:Tryptophan; D006851:Hydrochloric Acid