Therese M. Cotton

Immunoassay employing surface-enhanced Raman spectroscopy☆

Surface-enhanced Raman scattering (SERS) was used to measure binding between biomolecules with mutual affinity, including antigen-antibody interactions. The conjugation of nitro groups onto bovine serum albumin enhanced their specific SERS activity 10(4)-fold. A dye, 2-[4-hydroxyphenylazo]benzoic acid (HABA), with a major absorption at the Raman excitation frequency, demonstrated surface-enhanced resonance Raman scattering (SERRS) when captured from solution by avidin-coated silver films. Individual peak intensities showed a logarithmic relationship to the HABA concentration in solution over the range 10(-8) to 10(-5) M. Another resonance dye, p-dimethylaminoazobenzene (DAB) was covalently attached to an antibody directed against human thyroid stimulating hormone (TSH), without loss of antibody activity. The resultant conjugate was used in a sandwich immunoassay for TSH antigen: silver surfaces coated with anti-TSH antibody captured TSH antigen which in turn captured the DAB-anti-TSH antibody conjugate. A linear relationship was observed between the intensity of the resultant SERRS signals and the TSH antigen concentration over a range of from 4 to 60 microIU/ml. These results demonstrate the potential utility of the SERRS effect as a readout in a one-step, no wash immunoassay system.

Surface enhanced resonance Raman scattering (SERRS) as a probe of the structural differences between the Pr and Pfr forms of phytochrome

Surface enhanced resonance Raman scattering (SERRS) spectra have been obtained from the active, far‐red light absorbing (Pfr) and biologically inactive (Pr) forms of phytochrome adsorbed on silver colloids. Substantial differences between the SERRS spectra of the two forms in the low and high wavenumber regions are observed using 406.7 nm wavelength excitation. These differences reinforce those seen with 413.1 nm wavelength excitation in the high wavenumber region. Simultaneously, extensive differences are observed in the SERRS obtained from the same form in the low wavenumber region using 406.7 nm, as compared with 413.1 nm wavelength excitation. The relative intensity differences observed for the two forms, and those obtained using two slightly different excitation wavelengths to illuminate the same form, suggest that some type of subtle, proteincontrolled structural variation is responsible for the spectroscopic differences. A Z → E isomerization during the Pr → Pfr phototransformation is consistent with the SERRS data, although the overall chromophore conformations are most likely conserved for the native Pr‐ and Pfr‐phytochrome species. Slight out‐of‐plane ring twisting, accompanying the Pr → Pfr photoisomerization, may be responsible for the large difference in the spectroscopic properties of the native Pr and Pfr chromophores.

A normal coordinate analysis of the vibrational modes of the three redox forms of methylviologen: Comparison with experimental results

Abstract A normal coordinate analysis of the three redox forms of methylviologen (MV) was accomplished using a simplified valence force field. The calculated band frequencies are compared with those observed experimentally. The plausibility of the assignments for the Raman A g modes, as inferred from the normal coordinate analysis, is discussed. The assignments of several Raman A g modes were checked by a comparison of the observed Raman spectra of viologens with different substituents, such as methyl group deuterated methyl viologen (DV), N -octyl- N ′-methyl viologen (C 8 MV), N -hexadecyl- N ′-methyl viologen (C 16 MV) and dibenzyl viologen (BV), with MV. The values of the root mean square (r.m.s.) amplitudes of vibrations between the bonded and the nonbonded atom pairs, and r.m.s. Cartesian displacements from the equilibrium geometry for MV 2+ were computed. These data provide important insight with respect to the molecular geometry.

A surface-enhanced raman signal associated with functional manganese in oxygen-evolving photosystem II membranes

O2 evolution Manganese Photosystem II 33‐kDa protein 34‐kDa protein Photosynthesis

Surface-enhanced Raman scattering spectroscopy: Probing the lumenal surface of Photosystem II membranes for evidence of manganese

A surface-enhanced Raman scattering (SERS) peak at 225 cm− was reported in detergent-extracted, O2-evolving Photosystem II (PS II) appressed membrane fragments from spinach that were treated with CaCl2 (Seibert, M. and Cotton, T. (1985) FEBS Lett. 182, 34–38). CaCl2 removes the 17-, 23-, and 33-kDa extrinsic proteins associated with O2 evolution from the surface of the membrane without extracting functional Mn. The same peak has now been observed in PS II-enriched membrane fragments from Scenedesmus obliquus. The signal is related to the presence of Mn in spinach PS II membranes but not to the presence of Ca, Mg or Fe. Substitution of Br− for Cl− in the membranes without displacing Mn shifts the peak from 225 cm−1 to 140 cm−1. However, this shift is too great for the source of the SERS signals to be explained by simple Mn-Cl or Mn-Br vibrational modes. Evidence in addition to that presented in the previous publication indicates that the 33-kDa protein covers that part of the membrane that is the cause of the SERS signal. Due to altered properties of the SERS signal in the LF-1 mutant of Scenedesmus, which is affected in the D1 protein and binds less Mn than the wild type, we suggest that the 33-kDa protein shields a Mn-binding site on the D1 protein. Possible sources of the signal include (1) an Mn-O or Mn-N vibrational mode modified by chloride not present in the first coordination sphere; (2) a cooperative effect associated with the protein environment around the Mn-binding site that changes with the presence of halide; or (3) a species associated primarily with the Ag electrode surface that changes depending on the presence or absence of Mn and halide on the membrane surface. This last possibility might explain some of the results obtained with Br-treated spinach and Ca-treated Scenedesmus LF-1 mutant membranes.

A voltammetric study of asymmetric viologens in an organic solvent, an aqueous solution and in vesicle systems

Abstract Voltammetric studies of the asymmetric viologens, N-octyl-N-methyl viologen (C8MV), N-dodecyl-N-methyl viologen (C12MV) and N-hexadecyl-N-methyl viologen (C16MV), were performed using a glassy carbon electrode in acetonitrile, an aqueous solution and vesicle systems. For comparison, the cyclic voltammograms of the symmetric viologen, N,N-dimethyl viologen (MV) are also presented. In the organic solvent, the voltammograms of all the viologens are nearly identical. However, in aqueous solutions, the viologen chainlength has a large effect on the cyclic voltammetric (CV) behavior. Major differences were observed between the viologens in aqueous solutions and in vesicle systems. These observations are attributed to hydrophobic and electrostatic interactions between the viologen and the vesicle. In addition, the extent of the interaction between a particular viologen with vesicles was found to vary for different redox states. This finding was used to explain some unusual phenomena.

Spectroscopic analysis of chlorophyll model complexes: methyl ester ClFe(III)pheophorbides

As models for chlorophyll a (Chl a), methyl ester ClFe(III)pheophorbides (1, pheophorbide a; 2, mesopheophorbide a; and 3, mesopyropheophorbide a) were examined by Fourier transform infrared (FTIR) absorption and resonance Raman (RR) spectroscopy. The infrared (IR) chlorin band above 1600 cm-1, assigned as a Ca-Cm mode (Andersson et al. (1987) J. Am. Chem. Soc. 109, 2908-2916) is shown to be metal-sensitive and responsive to spin state and coordination number for dihydroporphyrins, as well as being diagnostic for the chlorin vs. porphyrin or bacteriochlorin macrocycle. Frequency variations for this metallochlorin IR band thus parallel those of the v10 RR mode of porphyrins in their predictive utility. Qy excitation SERRS spectra of Chl a were compared with Qy excitation RR spectra of 1 and methyl Ni(II)pyropheophorbide a. The data demonstrate that 5-coordinate ClFe(III)pheophorbides are better models for chlorophylls than are ruffled 4-coordinate Ni(II)pheophorbides. Major spectral differences between the three chlorophyll models are associated with the C-9 keto and/or C-10 carbomethoxy vibrational modes. The approx. 1700 cm-1 IR band was formerly assigned solely to v(C = O) of the C-9 keto group. However, this IR feature shifts down to approx. 1685 cm-1 and nearly doubles in intensity when the C-10 carbomethoxy is removed, as for 3. Similar frequency downshifts coupled with intensity increases in the IR are found in the literature on chlorophylls. RR spectra of pheophorbides having the C-10 carbomethoxy group (1 and 2) have bands at both approx. 1700 and approx. 1735 cm-1. However, the C-9 keto v(C = O) mode of pyrophorbins also downshifts to approx. 1685 cm-1, as in the IR spectra. The approx. 1735 cm-1 ester RR mode disappears in the case of pyrophorbins, and is never RR active for nonconjugated esters of porphyrins or chlorins. These data demonstrate an interaction between the C-10 and C-9 carbonyls of phorbins. They also indicate that phorbins tend toward conjugation of the C-10 ester. Biological examples of such conjugation effects have recently been reported, e.g., for the Chl a pi-cation radical (Heald et al. (1988) J. Phys. Chem. 92, 4820-4824). Because the phorbin E ring is the major structural feature distinguishing chlorophylls from non-photosynthetic systems, the participation of the C-10 ester in ring conjugation is suggestive of its biological importance.

A comparative study of the resonance Raman spectra of bacterial cytochromes

Resonance Raman spectra were obtained for two newly isolated bacterial cytochromes, Alcaligenes faecalis (ATCC 8750) c554 and Alcaligenes faecalis c556. Their spectra were compared with those of mammalian cytochrome c and two other bacterial cytochromes, Paracoccus denitrificans c550 and Pseudomonas aeruginosa c551. The positions of the Raman bands indicated that, while Al. c554 and Al. c556 were c-type cytochromes with two thioether linkages, several common features found in their Raman spectra were anomalous. These features suggest that the electronic charge density of the porphyrin macrocycle of Al. c554 and Al. c556 is more asymmetric than that of other bacterial and mammalian c-type cytochromes. The Raman evidence indicates that the electronic properties of the heme are controlled by the protein in these two Alcaligenes cytochromes.

Surface-Enhanced Resonance Raman Scattering from Cytochromes Cand P-450 on Bare and Phospholipid-Coated Silver Substrates

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) have demonstrated that the native integrity of this heme-containing protein is preserved in the presence of citrate-reduced sols. Cyt c SERRS spectra exhibit similar spin and oxidation markers as the resonance Raman scattering (RRS) signals displayed by the native protein solution. Previous SERRS investigations of cyt c showed that the protein denatures at the substrate surface. This hemoprotein denaturation was attributed to an electrostatic interaction between the porphyrin and the SERRS-active substrate. The results presented in this study suggest that the citrate ions prevent the biomolecule from directly interacting with the silver-sol surface. The native state of cyt c is also retained with the addition of phosphatidylcholine and/or phosphate buffer. Although the SERRS bandshifts correlate with the RRS spectra, the relative intensities of specific cyt c frequencies change in the presence of adsorbed citrate, phosphate buffer, and phosphatidylcholine. These compounds may influence the orientation of the protein at the substrate surface, which would account for the spectral intensity differences. In order to study the orientation effects further, additional studies of cyt c adsorbed onto silver electrodes and thin silver films were conducted. A comparison of the SERRS relative band intensities and frequency bandshifts shows that the spectra of the protein adsorbed onto phospholipid-coated substrates closely correspond to the RRS cyt c solution spectra. In contrast to the behavior exhibited by cyt c, cytochrome P-450 (cyt P-450) adsorbed onto a phosphatidylcholine silver-sol substrate leads to a low to high spin-state conversion. The shifts in the spin-state markers are ascribed to a strong interaction of the cyt P-450 with the phospholipid coating.

Low Temperature Surface-Enhanced Resonance Raman Scattering (SERRS) From Cytochromes

Surface-enhanced resonance Raman scattering (SERRS) studies of cytochrome c (cyt c) and cytochrome P450 (cyt P450) as a function of laser irradiation time have demonstrated that the proteins are extremely sensitive to photodegradation. The results suggest that previous SERRS reports of hemoprotein denaturation on Ag surfaces may reflect photosensitivity rather than an effect of the protein-surface interaction. Photodamage was eliminated by submersion of the electrode into liquid nitrogen. This procedure resulted in stable SERRS spectra, even with prolonged irradiation. The use of a diode array detector also substantially reduces the laser exposure period ( < 1 minute) required to observe SERRS spectra of the protein. The application of low temperature SERRS spectroscopy to the study of substrate binding in P450b provided evidence for spin state conversion in the presence of substrate.