Kwok To Yue

Raman Spectroscopic Characterization of Human Breast Tissues: Implications for Breast Cancer Diagnosis

Development and application of laser-based diagnostic and therapeutic procedures have been hindered by the current technical inadequacies in tissue diagnosis and characterization. It is now possible to apply the techniques of Raman spectroscopy to achieve rapid, noninvasive, and nondestructive differentiation of diseased from normal tissues. Normal and diseased breast tissues were examined by Raman spectroscopy. The Raman spectra obtained contain features that are attributable to various amounts of carotenoids and lipids. A small contribution from a heme-type signal was detected in some samples of clinically abnormal yet histopathologically benign breast tissue, while a much stronger heme-type signal was detected in most of the breast cancers. Raman spectra of diseased breast tissue (benign and malignant) also show markedly diminished to absent contributions from lipids and reduced contributions from carotenoids. This laser-based spectroscopic modality is readily adaptable to reflected light microscopy and optical fiber techniques, making it potentially useful as an aid in real-time diagnosis, and may thus find application in the fields of histopathology and interventional radiology.

Effect of N-alkyl substituents on the DNA binding properties of meso-tetrakis (4-N-alkylpyridinium-4-yl)porphyrins and their nickel derivatives

Resonance Raman, NMR, and visible spectroscopies, as well as viscosity and equilibrium dialysis studies were used to assess the effect of the N-alkyl substituent of meso-tetrakis(4-N-alkylpyridinium-4-yl)porphyrin cations on DNA binding. The DNAs studied include the native DNA, calf thymus DNA (CT DNA), the synthetic polynucleotides [poly(dGdC)]2 and [poly(dAdT)]2, and the oligonucleotide d(TATACGTATA)2. Both the porphyrins and the metalloporphyrins containing Ni(II) were examined with the N-alkyl = propyl (TPrpyP(4) and NiTPrpyP(4)) and 2-hydroxyethyl (TEtOHpyP(4) and NiTEtOHpyP(4)). The results were compared to those from the parent porphyrins with the N-methyl substituent (TMpyP(4) and NiTMpyP(4)). For almost all the comparisons made, the new porphyrin cations gave results very similar to those for the TMpyP(4) species. The resonance Raman study indicated that for the three DNA polymers all the Ni species were in the four-coordinate form when bound to all three polymers. It is suggested that both TPrpyP(4) and TEtOHpyP(4) bind to GC regions of DNA in the same intercalative manner as TMpyP(4) with the N-alkyl substituent extended into the solvent. For AT regions of DNA, the binding of TPrpyP(4) and TEtOHpyP(4) is nonintercalative, as found previously for TMpyP(4). The NiPrpy(4) and NiTEtOHpyP(4) cations bind to these polymers in a similar manner to the apo-porphyrins. The similar Raman spectral changes for the three Ni porphyrins upon addition of [poly(dAdT)]2 suggest that partial intercalation is not occurring because models indicate that it would be difficult to accommodate the bulkier N-alkyl substituents.

The determination of the pKa of histidine residues in proteins by Raman difference spectroscopy

Sensitive Raman difference spectroscopy was used to monitor the protonation and deprotonation of histidine residues in apo-transferrin. We have shown previously that the behavior of small molecules and/or small molecular groups bound to proteins or other large macromolecules can be studied by Raman difference spectroscopy (Yue, K.T. et al. (1989) J. Raman Spectrosc. 20, 541-545). Using this method, we have measured the Raman difference spectra of human transferrin at different pH values with respect to pH 8.9, titrating its various histidine residues. About 12 +/- 2 of the 19 residues were titrated. The pH difference spectrum of transferrin obtained is very similar to that of histidine in solution, but with clear differences in the 1200-1400 cm-1 region. A titration curve with pKa of 6.08 +/- 0.01 fit the data of histidine in solution and a value of 6.56 +/- 0.02 was found for the average value of the 12 histidine residues inside transferrin. The technique has enough sensitivity at present to monitor a single histidine residue in a 130 kDa molecule and to determine the titration curve of one residue in a 40 kDa protein.

Methoxypsoralen phototherapy oftransitional cell carcinoma

Abstract Objectives. The goal of this research was to assess whether methoxypsoralencompounds in combination with ultraviolet light were effective in preventing cellular proliferation in an in vitro model of human transitional cell carcinoma. Methods. Three methoxypsoralen compounds, 5-methoxypsoralen (5-MOP), 8-methoxypsoralen (8-MOP), and 4′-aminomethyl 4,5′-8′-trimethylpsoralen (AMT), were added in vitro to T-24 transitional cell carcinoma cells. Psoralens directly bind to DNA, cross-linking the strands when exposed to ultraviolet light and thereby prevent cellular division. Results. In vitro activity was demonstrated utilizing AMT and ultraviolet radiation at 320 to 340 nm, preventing cellular proliferation in T-24 transitional cell carcinoma. Conclusions. Methoxypsoralen compounds in combination with ultraviolet light are effective in preventing proliferation of bladder carcinoma cells in vitro. This therapy may prove to be effective in clinical early stage transitional cell carcinoma and warrants further assessment.

X-ray absorption and resonance raman spectroscopy of human myeloperoxidase at neutral and acid pH

Myeloperoxidase (MPO), an important enzyme in the oxygen-dependent host defense system of human polymorphonuclear leukocytes, utilizes hydrogen peroxide to catalyze the production of hypochlorous acid, an oxidizing bactericidal agent. While MPO shows significant sequence homology with other peroxidases and this homology is particularly striking among the active-site residues, MPO exhibits unusual spectral features and the unique ability to catalyze the oxidation of chloride ions. We have investigated the MPO active-site with X-ray absorption (XAS) and resonance Raman (RRS) spectroscopies at neutral pH and also at the physiological acidic pH (pH approximately 3) and have compared these results with those of horseradish peroxidase (HRP). At pH 7.5, XAS results show that the iron heme active site is 6-coordinate where the distal ligand is likely nitrogen or oxygen, but not sulfur. The heme is distorted compared to HRP, other peroxidases, and heme compounds, but at pH approximately 3, the distal ligand is lost and the heme is less distorted. RRS results under identical pH conditions show that the skeletal core-size sensitive modes and v3 are shifted to higher frequency at pH approximately 3 indicating a 6- to 5-coordination change of high spin ferric heme. In addition, a new band at 270 cm(-1) is observed at pH approximately 3 which is consistent with the loss of the sixth ligand. The higher symmetry of the heme at pH approximately 3 is reflected by a single v4 mode in the (RRS) spectrum. HRP also loses its loosely associated distal water at this pH, but little change in heme distortion is observed. This change suggests that loss of the distal ligand in MPO releases stress on the heme which may facilitate binding of chloride ion.

Direct evidence of the metal-free nature of sirohydrochlorin in desulfoviridin

We have obtained direct evidence that the majority of the sirohydrochlorin chromophore in the dissimilatory sulfite reductase desulfoviridin from Desulfovibrio gigas, is not associated with any metal. The evidence comes from resonance Raman measurements of native and deuterated samples of desulfoviridin. The breathing mode v4 (or v4*) at 1336 cm-1 in the native enzyme is downshifted to 1326 cm-1 upon deuteration. This mode is not sensitive to deuteration if a metal is present at the center of the chromophore inside protein or in solution. The results also establish the existence of exchangeable core hydrogen(s) at the pyrrolic nitrogen(s).

Simple, Versatile Liquid Nitrogen Cryostat for Raman Studies:

Resonance Raman studies can often benefit from decreased sample temperature. With biological samples like frozen protein solutions, the spectral peaks are significantly narrowed at liquid nitrogen temperature due to the reduced molecular motion. Lowering the temperature can also minimize protein denaturation from local heating by the exciting laser beam. Semiconductors and their superlattices are another class of materials where Raman measurements often must be made at low temperatures. For many such samples the Raman features are very weak or not observable at room temperature; some mode intensities have a strong temperature dependence, and some modes are active only at low temperature. It is significant that most, if not all, of the features can be observed at liquid nitrogen temperatures, even though published reports often use lower temperatures requiring the more expensive and cumbersome liquid helium.

Resonance Raman study of sirohydrochlorin and siroheme in sulfite reductases from sulfate reducing bacteria

Soret-excited resonance Raman (RR) spectra are reported for the sirohemes in the oxidized and Cr11(EDTA)-reduced forms of both desulforubidin from D. baculatus (DSR) and the low molecular weight sulfite reductase from D. vulgaris (1SIR) and for sirohydrochlorin in the oxidized form of desulfoviridin from D. gigas (DSV). Several patterns in the RR spectra of these enzymes can be utilized as signatures for the siroheme/sirohydrochlorin moiety. The active site for DSR and 1SIR consists of a siroheme exchange-coupled to a [4Fe-4S]2+ cluster. Upon addition of Cr11(EDTA), the active center of DSR and 1SIR undergoes a one-electron and two-electron reduction, respectively. The RR spectra of DSR suggest that the siroheme iron is high spin and 5-coordinate in the oxidized enzyme and probably remains high spin and 5-coordinate upon reduction. The iron in the siroheme of oxidized 1SIR changes from a low spin and probably 6-coordinate configuration to a high spin, 5-coordinate complex upon two-electron reduction of the active site. Close similarities between the RR spectral features of the two-electron-reduced assimilatory sulfite reductases from E. coli and from D. vulgaris (1SIR) are discussed.

RESONANCE RAMAN STUDY ON THE IRON-SULFUR CENTERS OF DESULFOVIBRIO GIGAS ALDEHYDE OXIDOREDUCTASE

Resonance Raman spectra of the molybdenum containing aldehyde oxidoreductase from Desulfovibrio gigas were recorded at liquid nitrogen temperature with various excitation wavelengths. The spectra indicate that all the iron atoms are organised in [2Fe-2S] type centers consistent with cysteine ligations. No vibrational modes involving molybdenum could be clearly identified. The features between 280 and 420 cm-1 are similar but different from those of typical plant ferredoxin-like [2Fe-2S] cluster. The data are consistent with the presence of a plant ferredoxin-like cluster (center I) and a unique [2Fe-2S] cluster (center II), as suggested by other spectroscopic studies. The Raman features of center II are different from those of other [2Fe-2S] clusters in proteins. In addition, a strong peak at ca. 683 cm-1, which is not present in other [2Fe-2S] clusters in proteins, was observed with purple excitation (406.7-413.1 nm). The peak is assigned to enhanced cysteinyl C-S stretching in center II, suggesting a novel geometry for this center.

Raman Difference Spectroscopy And The Energetics Of Enzymatic Catalysis

Typically, there are specific molecular interactions between a substrate and enzyme, which occur during enzymatic catalysis, that must be sufficient to not only reduce the transition state barrier appropriate for the reaction but also define a particular (usually small) set of chemical reactions. Both the origins and strengths of these interactions are fundamental issues in understanding how enzymes work. While more structural information is increasingly available from X-ray crystallographic studies, the extent of these interactions and the electronic character of the substrate and nearby protein groups within the active site generally must be simply surmised from the structural data and kinetic studies. Rarely are these molecular properties directly measured. We have approached this problem by determining the vibrational spectra of bound substrates using Raman spectroscopy. The observed vibrational frequencies are a measure of force constants between particular atoms, and these constants can be related in turn to bond orders and electronic distributions between these atoms.