Quantitative analysis of nucleic acids, proteins, and viruses by Raman band deconvolution

Abstract

A constrained, iterative Fourier deconvolution method is employed to enhance the resolution of Raman spectra of biological molecules for quantitative assessment of macromolecular secondary structures and hydrogen isotope exchange kinetics. In an application to the Pf1 filamentous bacterial virus, it is shown that the Raman amide I band contains no component other than that due to alpha-helix, indicating the virtual 100% helicity of coat proteins in the native virion. Comparative analysis of the amide I band of six filamentous phages (fd, If1, IKe, Pf1, Xf, and Pf3), all at the same experimental conditions, indicates that the subunit helix-percentage ranges from a high of 100% in Pf1 to a low of 71% in Xf. Deconvolution of amide I of Pf3 at elevated temperatures, for which an alpha-to-beta transition was previously reported (Thomas, G. J., Jr., and L. A. Day, 1981, Proc. Natl. Acad. Sci. USA., 78:2962-2966), allows quantitative evaluation of the contributions of both alpha-helix and beta-strand conformations to the structure of the thermally perturbed viral coat protein. Weak Raman lines of viral DNA bases and coat protein side chains, which are poorly resolved instrumentally, are also distinguished for all viruses by the deconvolution procedure. Application to the carbon-8 hydrogen isotope exchange reaction of a purine constituent of transfer RNA permits accurate determination of the exchange rate constant, which is in agreement with calculations based upon curve-fitting methods.

Macroscale Plasmonic Substrates for Highly Sensitive Surface‐Enhanced Raman Scattering

“…These parameters enable the use of the portable handheld Raman system for macroscopic measurements. Deconvolution of the bands at 1516 and 1552 cm À1 (Figure 4 A) [19] and plotting of the band-area ratio against the CO concentration gave a linear correlation, with an impressive R 2 value of 0.9917 (Figure 4 B). This result demonstrates the quantitative nature of this method of analysis.…”

Quantitative prediction of protein secondary structure —Where is the lacuna?

“…Finally, it appears that there is considerable merit in trying to get a quantitative picture of the secondary structures of proteins from the Amide I and IIl bands in the laser-Raman spectra by correlating intensity changes with the dinedral angles tb and ~, which change with the secondary structures (24,35). For band-fitting spectral profiles, the constrained deconvolution technique may be more suitable (23).…”

A systematic approach toward the analysis of drug–DNA interactions using Raman spectroscopy: The binding of metal‐free bleomycins A 2 and B 2 to calf thymus DNA

“…16 In this investigation, conventional Raman spectroscopy was utilized to investigate the nature of binding of the Blm BIT moiety with calf thymus DNA (ct-DNA). Calf thymus DNA has been extensively characterized by Raman and ir spectroscopy [17][18][19][20][21][22][23][24][25][26][27][28][29][30][31][32] and hence makes an ideal native mammalian DNA system for the purpose of studying drug-DNA interactions. The clinical mixture of Blm A 2 and B 2 was employed as this is the substance that is normally administered in vivo to cancer patients.…”

“…12,18,20 The application of curvefitting techniques to normal Raman spectra represents another way to examine drug-DNA interactions. [23][24][25][26][27][28][29] In this study, a curvefitting technique has been employed in order to systematically analyze any spectral perturbations in order to detect any frequency shifts that may occur as well as changes in band intensity. The present study shows extensive alterations in the intensities of the bands in the Raman spectrum in the region 1160 -1500 cm Ϫ1 upon binding of metal free Blm.…”

“…This band, assigned to PO 2 Ϫ symmetric stretch, has been used repeatedly by workers in the field as an intensity standard. 17,19,[23][24][25][26][27][28] This normalization was done after baselines were corrected using software in Spectracalc@ (Galactic Industries). All files were corrected using the same baseline parameters so that these corrections were consistent for each spectrum before adding individual files from individual scans together.…”