Nai-Teng Yu

Laser Raman spectroscopy and the conformation of insulin and proinsulin

Abstract Laser-excited Raman spectra of native and denatured insulin in the solid state and aqueous solution are reported. Striking spectral changes on denaturation have been observed in the following spectral regions: (a) the amide I and III, (b) the SS and CS stretching, (c) the skeletal bending and (d) skeletal stretching region. Detailed analysis of these spectral changes indicates that extensive conformational changes have taken place in the conversion of native to denatured fibrous insulin and that fibrous insulin exists in a β-conformation as proposed by Ambrose & Elliott (1951). Comparisons have also been made between the spectra of native insulin in crystals and in solution at various pH values. Small spectral differences were observed and interpreted in terms of conformational changes and ionization effect. We also present the first Raman spectrum of proinsulin in the solid state and compare it to that of insulin. A total of eleven lines, known to be conformation-dependent, was found to be in good agreement between these two spectra. This strongly suggests that the insulin moiety of proinsulin exists in a conformation very nearly the same as insulin itself, which is consistent with the conclusions of Frank & Veros (1968). The differences between these two spectra are attributed to the connecting peptide, characteristic of its conformation. A graphical subtraction between these two spectra in the amide I region shows a peak at 1663 cm −1 with a shoulder near 1685 cm −1 . These two frequencies are indicative of α-helical structure and random-coil form, respectively, in the C-peptide.

Laser raman spectra of oxidized hydroperoxidases

Resonance Raman spectra of oxidized hydroperoxidases are examined for shifts in the structure-sensitive, anomalously polarized bands; these are found, respectively, at 1576, 1567 and 1570 cm-1 in the high-spin resting enzymes: horse radish peroxidase, horse blood catalase, and cytochrome c peroxidase. In compound II of horse radish peroxidase and horse blood catalase, and in the enzyme-substrate complex of cytochrome c peroxidase, this band appears at 1587-1590 cm-1 and indicates the iron atom is now in-plane with the porphyrin ring. Weak Raman scattering found with horse radish peroxidase I is consistant with a porphyrin eta-cation radical formulation.

A preliminary Raman spectroscopic study of native zinc-insulin crystals

Abstract A Raman spectrum of rhombohedral zinc-insulin crystals is reported and partially interpreted. One interesting finding is that the intrachain disulfide bond, A6-11, may have a local geometry different from that of the interchain disulfide bonds, B7-A7 and B19-A20. Another finding is that the amide III region consists of three resolved peaks at 1239, 1270 and 1288 cm −1 , indicating the existence of three distinct structural components in the polypeptide chains of the insulin crystals. These interpretations are compared with the X-ray crystallographic results of Hodgkin and co-workers.

Light absorption, electron paramagnetic resonance and resonance Raman characteristics of nitridochromium(V) protoporphyrin-IX and its reconstituted hemoproteins.

A surprisingly stable complex of the photolyzed product of azidochromium(III)protoporphyrin-IX was prepared and examined by light absorption, electron paramagnetic resonance (EPR) and resonance Raman spectroscopies. The characteristic EPR spectrum for this complex was consistent with a nitridochromium(V)-porphyrin complex which was two oxidation equivalents above the resting Cr(III) complex. The Cr(V)-N stretching mode was observed at 1010 cm-1 by resonance Raman spectroscopy. A simple diatomic harmonic oscillation model gave a force constant of 6.7 mdyn/A for the Cr(V)-N bond, in the region characteristic for the metal-nitrogen triple bond. Nitridochromium(V) protoporphyrin-IX reconstituted myoglobin and cytochrome c peroxidase were prepared for the first time. The nitridochromium(V)-porphyrins in these apo-proteins were unstable in contrast with the protein-free chromium(V)porphyrin. Upon irradiation of the azide complexes of the chromium(III) protoporphyrin-IX reconstituted myoglobin and cytochrome c peroxidase with ultraviolet light aerobically at room temperature, the characteristic optical and EPR spectra for nitridochromium(V) derivatives were observed. The optical spectra of these photo-induced products were different from those of the nitridochromium(V) protoporphyrin-IX reconstituted hemoproteins. The electrochemical structures of the unusual metalloporphyrin seemed to be modulated by the heme surrounding amino acid residues.