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.
Song
S H
SH
Department of Chemistry, University of Pittsburgh, Pennsylvania 15260.
Asher
S A
SA
D002101:Cacodylic Acid / Q000737:chemistry; D006801:Humans; D007501:Iron / Q000737:chemistry; D008706:Methemoglobin / Q000737:chemistry*; D010472:Perchlorates; D064586:Selenic Acid; D012643:Selenium / Q000737:chemistry; D018036:Selenium Compounds; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013431:Sulfates / Q000737:chemistry
D010472:Perchlorates; D018036:Selenium Compounds; D013431:Sulfates; D008706:Methemoglobin; D002101:Cacodylic Acid; D007501:Iron; D012643:Selenium; D064586:Selenic Acid