INTERNAL INTENSITY STANDARDS FOR HEME PROTEIN UV RESONANCE RAMAN STUDIES - EXCITATION PROFILES OF CACODYLIC ACID AND SODIUM SELENATE

We examine the utility of SO4(2-), ClO4-, cacodylic acid, and SeO4(2-) as internal intensity standards for Raman spectral measurements of protein structure. We find that 0.1 M SO4(2-) and ClO4- perturb the protein tertiary structure of aquomethemoglobin (met-Hb) and its fluoride (met-HbF) and azide (met-HbN3) complexes. Changes occur for the tryptophan near-UV absorption bands, the iron spin state is altered, and the fluoride ligand affinity decreases. Concentrations of ClO4- and SO4(2-) as low as 0.1 M suppress the met-HbF quaternary R --> T transition induced by the allosteric effector inositol hexaphosphate (IHP). In contrast, similar concentrations of cacodylic acid and SeO4(2-) show little effect on the hemoglobin tertiary or quaternary protein structures or upon the R --> T transition induced by IHP. We measure the Raman cross sections of cacodylic acid and SeO4(2-) between 218 and 514.5 nm and find that for UV excitation they are ca. 5-fold larger than ClO4- or SO4(2-). Thus, cacodylic acid and selenate can be used at lower concentrations. Cacodylic acid and SeO4(2-) are superior Raman internal intensity standards for protein structural studies.