Xiaojie Zhao

SOLID-STATE TUNABLE KHZ ULTRAVIOLET LASER FOR RAMAN APPLICATIONS

Performance characteristics are described for a kilohertz solid-state laser source for resonance Raman spectroscopy. Narrow line excitation in the 205–230 nm region is provided by quadrupling a titanium: sapphire laser which is pumped by the second harmonic of a Q-switched YLF laser. The combination of tunability, narrow line, high average power, and good stability makes this laser suitable for ultraviolet resonance Raman (UVRR) applications. UVRR spectra of hemoglobin, excited at 229 nm, are as high in quality as those produced by continuous-wave intracavity frequency-doubled Ar+ laser excitation. Raman excitation profiles are reported for aromatic acids in aqueous solution and in hemoglobin.

TIME-RESOLVED RAMAN SPECTROSCOPY WITH A TUNABLE ULTRAVIOLET KILOHERTZ NANOSECOND LASER

Time-resolved resonance Raman (TR3) spectra are obtained with a pair of Q-switched Nd : YLF-pumped Ti : sapphire lasers, generating tunable (810–920 nm) ∼20 ns pulses at a 1 kHz repetition rate. Frequency doubling in lithium borate (LBO) provides blue (405–460 nm) pump and probe pulses, while UV probe pulses, tunable from 205 to 230 nm, can be generated by doubling the second harmonic in β-barium borate (BBO). Pump and probe pulse timing are controlled electronically. A timing sequence is implemented in which exposure of the multichannel detector alternates between positive and negative time delays between pump and probe pulses, so that accumulated difference spectra are free of artifacts from spectrograph drift or gradual decomposition of the sample. The system was tested on the carbonmonoxy hemoglobin (HbCO) photocycle, for which UV TR3 spectra have previously been reported. HbCO was pumped at 419 nm, at the maxima of the strong Soret absorption band, and saturation of the photoresponse (maximum deligation) was established by measuring the intensity ratio of the HbCO and deoxyHb ν4 porphyrin RR bands, generated with 425 nm probe pulses. UV TR3 difference spectra were obtained at time intervals from 0.06 to 20 µs using 229 nm probe pulses. They are in good agreement with those recorded previously with a pair of 300 Hz excimer-dye lasers. The time required to achieve a comparable signal-to-noise ratio was eight times shorter with the 1 kHz Nd : YLF-Ti : S lasers. Copyright

Subunit-Selective Interrogation of CO Recombination in Carbonmonoxy Hemoglobin by Isotope-Edited Time-Resolved Resonance Raman Spectroscopy

Hemoglobin (Hb) is an allosteric tetrameric protein made up of alphabeta heterodimers. The alpha and beta chains are similar, but are chemically and structurally distinct. To investigate dynamical differences between the chains, we have prepared tetramers in which the chains are isotopically distinguishable, via reconstitution with (15)N-heme. Ligand recombination and heme structural evolution, following HbCO dissociation, was monitored with chain selectivity by resonance Raman (RR) spectroscopy. For alpha but not for beta chains, the frequency of the nu(4) porphyrin breathing mode increased on the microsecond time scale. This increase is a manifestation of proximal tension in the Hb T-state, and its time course is parallel to the formation of T contacts, as determined previously by UVRR spectroscopy. Despite the localization of proximal constraint in the alpha chains, geminate recombination was found to be equally probable in the two chains, with yields of 39 +/- 2%. We discuss the possibility that this equivalence is coincidental, in the sense that it arises from the evolutionary pressure for cooperativity, or that it reflects mechanical coupling across the alphabeta interface, evidence for which has emerged from UVRR studies of site mutants.

UV resonance Raman probe of heme-bound imidazole established by15N-labeling of hemoglobin

Recent studies of Cu, Zn superoxide dismutase, and of zinc-finger peptides have established that histidine ligands can be detected in ultraviolet resonance Raman (UVRR) spectra, following NH/D exchange of the imidazole. UVRR spectroscopy therefore offers promise for monitoring histidine ligation in heme proteins. In this work, we characterize heme-bound histidine UVRR bands for N-acetyl-microperoxidase-8 (MP-8) and microperoxidase-11 (MP-11), and also for hemoglobin (Hb). The Hb UVRR spectra are dominated by tyrosine and tryptophan contributions, but a band appears at 1340 cm−1 in D2O solution, which is assigned to a mode of Fe-bound imidazole. This band shifted 24 cm−1 in protein which was labeled with 15N via expression of the Hb gene in E. coli grown on 15NH4+. In MP-11, the position of this band is insensitive to ligation or oxidation state changes, but it is 2 cm−1 lower in deoxyHb than in the CO adduct. This shift may reflect mechanical forces on the proximal histidine in the T state, and/or changes in its H-bonding.

Characterization of DNA isolated from normal and cancerous ovarian tissues by ultraviolet resonance Raman spectroscopy

We report significant differences in UV resonance Raman (UVRR) spectra of DNA samples from normal and cancerous tissues. The four bases of DNA, adenosine, thymine, guanosine and cytidine, can be enhanced in UVRR spectra, and their intensities are very sensitive to base stacking and DNA H-bonding. 14 DNA samples from patients at different stages of ovarian cancer, 5 from normal, 2 from primary, 3 from metastasis primary and 4 from distant metastasis tumor tissues, were characterized by 257, 238, 229, 220 and 210 nm-excited UVRR spectra. Raman spectral difference between normal and tumor DNA could be readily detected.