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Cacimar Ramos ÁlvarezCentral New York – Puerto Rico AGEP
Universidad de Puerto Rico, Mayagüez
Setting up a femtosecond absorption pump/probe experimental setup for biomolecules analysis. The system consist of a commercially Ultrafast laser assemble form Spectra Physics, coupled with a “3D approach” detection system for sample analysis. The laser assemble is composed of a diode green laser (Millenia) used to pump a Ti-Sapphire oscillator (Tsunami). This beam is directed to a regenerative amplifier that is been pumped with a YLF laser (Merlin). The output of the regenerative amplifier has a time resolution of approximately 100 fs, with 1 KHz repetition rate and a power of 800 mj. Recent work has been directed on the installation of an ICCD (Intensifed Charge Couple Device) for the detection of the absorption signals from the sample. This new setup will make possible the acquisition of range of wavelength instead of single wavelength as previously done using a PMT detector. We will then be able to generate a 3D surface that presents the relationship between wavelength, time and signal absorption. The software needed to couple the detector with the optical delay (wich gives us the time resolution of the experiments) has been “homemade” coded using Visual Basic. The sample handling mechanism has also been developed and constructed in site in order to handle the samples and different ligand complexes. Various sample management approaches and schemes have been implemented and are available for the different samples and it’s handling requirements.
Our experiments will focus on the kinetic studies of hemoglobin’s from the local clam Lucina Pectinata. This clam posses three different heme proteins called HbI, HbII and HbIII, the first one capable of binding H2S in its active site. In order to elucidate the various mechanisms involved and how those are affected by the active site residues composition, mutants have been expressed and produced and will also been study on this new laser system setup. The new detection setup will provides the opportunity of applying global fitting techniques to the data in order to determine the different kinetic constant for the ligand complex under study.
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