258 research outputs found
Direct detection of a dioxygen adduct of cytochrome a3 in the mixed valence cytochrome oxidase/dioxygen reaction
No full textTime-resolved resonance Raman spectra have been recorded during the reaction of mixed valence (a3+ a32+) cytochrome oxidase with dioxygen at room temperature. In the spectrum recorded at 10 μs subsequent to carbon monoxide photolysis, a mode is observed at 572 cm-1 that shifts to 548 cm-1 when the experiment is repeated with 18O2. The appearance of this mode is dependent upon the laser intensity used and disappears at higher incident energies. The high frequency data in conjunction with the mid-frequency data allow us to assign the 572 cm-1 mode to the Fe-O stretching vibration of the low-spin O2 adduct that forms in the mixed valence cytochrome oxidase/dioxygen reaction. The 572 cm-1 ν(Fe2+-O2) frequency in the mixed valence enzyme/O2 adduct is essentially identical to the 571 cm-1 frequency was measured for this mode during the reduction of O2 by the fully reduced enzyme (Varotsis, C., Woodruff, W.H., and Babcock, G.T. (1989) J. Am. Chem. Soc. 111, 6439-6440; Varotsis, C., Woodruff, W.H., and Babcock, G.T. (1990) J. Am. Chem. Soc. 112, 1297), which indicates that the O2-bound cytochrome a3 site is independent of the redox state of the cytochrome a/Cu(A) pair. The photolabile oxy intermediate is replaced by photostable low- or intermediate-spin cytochrome a33+, with t( 1/2 ) ~ 200 μ
Study of maillard reactions with coupled chromatographic and spectroscopic techniques
No full textΗ παρούσα ερευνητική εργασία έχει ως στόχο τη μελέτη μοντέλων αντιδράσεων Maillard με συνδυασμό αναλυτικών τεχνικών, όπως η χρωματογραφία HPLC και η φασματοσκοπία UV-vis και ATR-FTIR. Η αντίδραση Maillard περιλαμβάνει ένα σύνθετο δίκτυο χημικών αντιδράσεων που εμφανίζονται σε τρόφιμα μετά από επεξεργασία σε υψηλές θερμοκρασίες. Ένας περαιτέρω σκοπός αυτής της μελέτης είναι η ανάπτυξη απλών φασματοσκοπικών μεθόδων εφαρμογής των φασματοσκοπικών ιδιοτήτων των συστατικών των τροφίμων.
Η αντίδραση της ασπαραγίνης και της φρουκτόζης σε υψηλή θερμοκρασία μελετήθηκε με την τεχνική σύζευξης της HPLC-ATR-FTIR. Η σύζευξη της υγρής χρωματογραφίας υψηλής απόδοσης (HPLC), με την ανίχνευση FTIR, παρέχει μια περαιτέρω δυνατότητα επιβεβαίωσης των αλλαγών του χημικού συστήματος σε μια νέα πειραματική διάταξη.
Επιπλέον, ο σχηματισμός των προϊόντων Amadori ταυτοποιήθηκε χρησιμοποιώντας μια συνδυασμένη αναλυτική τεχνική της Εκχύλισης Στερεάς Φάσης (SPE) και της φασματοσκοπίας υπέρυθρου εξασθενημένης ολικής ανάκλασης με μετασχηματισμό Fourier (ATR-FTIR).
Η απομόνωση των προϊόντων αντίδρασης της ασπαραγίνης με αναγωγικά σάκχαρα σε αλκαλικό pH και υψηλή θερμοκρασία ανιχνεύθηκε με συνδυασμό υγρής χρωματογραφίας υψηλής απόδοσης (HPLC), σε συνδυασμό με έναν συλλέκτη κλασμάτων. Τα προϊόντα αντίδρασης αναλύθηκαν με φασματοφωτομετρία ορατού-υπεριώδους (UV-vis) και υπέρυθρης ακτινοβολίας με μετασχηματισμό Fourier (FTIR). Τα φάσματα ορατού-υπεριώδους (UV-vis) και FTIR των απομονωθέντων προϊόντων αντίδρασης Maillard έδειξαν αλλαγές ευαίσθητες στη δομή όπως συσχετίζονται με απαμίνωση και σχηματισμό συζυγών ασπαραγίνης-σακχαρίτη. Η απόδειξη για τη σύμπλεξη ιόντων μετάλλων χαλκού Cu(II) με τα προϊόντα αντίδρασης Maillard υποστηρίζεται από τη φασματοσκοπία UV-vis και FTIR.
Το ίδιο μοντέλο συστήματος χρησιμοποιήθηκε για να διερευνηθεί η αλληλεπίδραση των προϊόντων αντίδρασης Maillard που σχηματίστηκαν από τις αντιδράσεις των προερχόμενων από σακχαρίτη ενώσεων, δημιουργώντας ενδιάμεσα με πρωτεΐνες, πιο συγκεκριμένα με αιμοσφαιρίνη και μυοσφαιρίνη. Παρατηρήθηκαν τα φασματικά γεγονότα κατά την προσθήκη διαφορετικών προϊόντων αντίδρασης Maillard (MRPs) σε αιμοσφαιρίνη και μυοσφαιρίνη. Αυτό οδήγησε στον σχηματισμό μιας τροποποιημένης πρωτεϊνικής μορφής, γνωστής ως αιμόχρωμα (hemichrome).
Επιπλέον, χρησιμοποιήθηκε φασματοσκοπία φθορισμού ως συμπληρωματική τεχνική για τη μελέτη της αντίδρασης της αιμοσφαιρίνης με τα προϊόντα αντίδρασης Maillard (MRPs) από ένα σύστημα μοντέλου ασπαραγίνης-μονοσακχαρίτη. Η τροποποίηση της αιμοσφαιρίνης από τα προϊόντα αντίδρασης Maillard (MRPs) απεδείχθη μέσω συγκεκριμένων αλλαγών στην τρυπτοφάνη στα φάσματα φθορισμού.The present research work was aimed to study model Maillard reactions by a combination of analytical techniques such as HPLC, UV-vis and ATR-FTIR spectroscopies. The Maillard reaction involves a complex network of chemical reactions that occur in food after processing at high temperatures. A further purpose of this study was to develop simple spectroscopic methods of application of the spectroscopic properties of the food constituents.
The reaction of asparagine and fructose at high temperature was studied by the coupling technique of HPLC-ATR-FTIR. The coupling of high performance liquid chromatography (HPLC) with FTIR detection provides a further capability for confirming chemical system changes in a new experimental layout.
Furthermore, the formation of the Amadori products was identified by employing a combined analytical technique of Solid Phase Extraction (SPE) and Attenuated Total Reflection-Fourier Transform Infrared Spectroscopy (ATR-FTIR).
The isolation of reaction products of asparagine with reducing sugars at alkaline pH and high temperature was probed by a combination of high performance liquid chromatography (HPLC) coupled with a Fraction Collector. The reaction products were analyzed by UV-vis and Fourier transform infrared (FTIR) spectrophotometry. The UV- vis and FTIR spectra of the isolated Maillard reaction products showed structure sensitive changes as depicted by deamination events and formation of asparagine saccharide conjugates. Evidence for Cu (II) metal ion complexation with the Maillard reaction products is supported by UV-Vis and FTIR spectroscopy.
The same model system was utilized to investigate the interaction of Maillard reaction products formed by the reactions of saccharide-derived compounds creating intermediates with proteins, more specifically with hemoglobin and myoglobin. The spectral events upon addition of discrete MRPs to hemoglobin and myoglobin were monitored. This led to the formation of a modified protein adduct known as a hemichrome.
Additionally, fluorescence spectroscopy was employed as a complementary technique to study the reaction of hemoglobin with MRPs from an asparagine-sugar model system. The modification of hemoglobin by MRPs was illustrated through tryptophan- specific changes in the fluorescent spectra.Complete
Probing Protonation/Deprotonation of Tyrosine Residues in Cytochrome ba3 Oxidase from Thermus thermophilus by Time-resolved Step-scan Fourier Transform Infrared Spectroscopy
No full textElucidating the properties of the heme Fe-Cu B binuclear center and the dynamics of the protein response in cytochrome c oxidase is crucial to understanding not only the dioxygen activation and bond cleavage by the enzyme but also the events related to the release of the produced water molecules. The time-resolved step-scan FTIR difference spectra show the ν 7a(CO) of the protonated form of Tyr residues at 1247 cm -1 and that of the deprotonated form at 1301 cm -1. By monitoring the intensity changes of the 1247 and 1301 cm -1 modes as a function of pH, we measured a pK a of 7.8 for the observed tyrosine. The FTIR spectral changes associated with the tyrosine do not belong to Tyr-237 but are attributed to the highly conserved in heme-copper oxidases Tyr-136 and/or Tyr-133 residue (Koutsoupakis, K., Stavrakis, S., Pinakoulaki, E., Soulimane, T., and Varotsis, C. (2002) J. Biol. Chem. 277, 32860-32866). The oxygenation of CO by the mixed-valence form of the enzyme revealed the formation of the ∼607 nm P (Fe(IV)=O) species in the pH 6-9 range and the return to the oxidized form without the formation of the 580 nm F form. The data indicate that Tyr-237 is not involved in the proton transfer pathway in the oxygenation of CO by the mixed-valence form of the enzyme. The implication of these results with respect to the role of Tyr-136 and Tyr-133 in proton transfer/gating along with heme a 3 ringD propionate-H 2O-ring A propionate-Asp-372 site to the exit/output proton channel (H 2O pool) is discusse
Σχέση δομής – λειτουργίας του φωτοσυνθετικού μηχανισμού των διατόμων Phaeodactylum Tricornutum και Thalassiosira
No full textDiatoms are unicellular photosynthetic organisms responsible for 20% of the photosynthesis that takes place on the planet. The FCPs (Fucoxanthin Chlorophyll Proteins) complexes consist of chlorophylls a/c and the carotenoids fucoxanthin and diadinoxanthin which are responsible for light harvesting. These organisms utilize photoprotection mechanisms to ensure their survival under intense and variable light conditions.
In this thesis, a combination of Uv-vis, resonance Raman and Fluorescence spectroscopy as well as the analytical technique of high-performance liquid chromatography are used to investigate the response of two diatom species under different growth conditions. The species Phaeodactylum Tricornutum has been selected from the group of pennate diatoms and Thalassiosira Pseudonana from centric diatoms. By altering the growth conditions, changes were observed in the concentration of the produced pigments in addition to significant differences in the properties and interactions they display. These changes seem to depend on both the intensity and the wavelength of the light source used for the growth of the diatoms.
In addition, isolation and study of the FCPs properties from both organisms has been carried out. Finally, by replacing 14N with the 15N isotope on the diatom Phaeodactylum Tricornutum, the observed N-sensitive vibrations using Raman spectroscopy were characterized.Τα διάτομα είναι μονοκύτταροι φωτοσυνθετικοί οργανισμοί υπεύθυνοι για το 20% της φωτοσύνθεσης που πραγματοποιείται στον πλανήτη. Για τη συλλογή φωτός επιστρατεύουν τα σύμπλοκα συλλογής φωτός FCPs (Fucoxanthin Chlorophyll Proteins) που αποτελούνται από τις χλωροφύλλες α και c καθώς και τα καροτενοειδή φουκοξανθίνη και διαδινοξανθίνη. Οι οργανισμοί αυτοί αξιοποιούν μηχανισμούς προστασίας έτσι ώστε να είναι εφικτή η επιβίωση τους σε μεταβαλλόμενα περιβάλλοντα όπως είναι οι αυξομειώσεις στην ένταση της ηλιακής ακτινοβολίας κατά τη διάρκεια της ημέρας.
Στην παρούσα διατριβή αξιοποιούνται οι φασματοσκοπικές τεχνικές απορρόφησης ορατού – υπεριώδους, φθορισμού και Raman καθώς και η αναλυτική τεχνική της υγρής χρωματογραφίας υψηλής απόδοσης για να μελετηθεί η απόκριση των κυττάρων δύο ειδών διατόμων κάτω από διαφορετικές συνθήκες ανάπτυξης. Από την κατηγορία των πτεροειδών διατόμων έχει επιλεγεί το είδος Phaeodactylum Tricornutum και από την κατηγορία των κεντρικών διατόμων το είδος Thalassiosira Pseudonana. Με την αλλαγή των συνθηκών ανάπτυξης παρατηρήθηκαν μεταβολές στη συγκέντρωση των παραγόμενων χρωμοφόρων μορίων καθώς και σημαντικές διαφορές στις ιδιότητες και τις αλληλεπιδράσεις που αυτά εμφανίζουν. Οι μεταβολές αυτές φαίνεται πως έχουν εξάρτηση τόσο από την ένταση όσο και από το μήκος κύματος της ακτινοβολίας που χρησιμοποιείται για την ανάπτυξη των διατόμων.
Επίσης έχει πραγματοποιηθεί απομόνωση και μελέτη των ιδιοτήτων των FCPs των συγκεκριμένων οργανισμών καθώς και ανάπτυξη του διατόμου Phaeodactylum Tricornutum με αντικατάσταση του 14Ν με το ισότοπο 15Ν με σκοπό τον χαρακτηρισμό των παρατηρούμενων N-ευαίσθητων δονήσεων με χρήση της φασματοσκοπίας Raman.Μέλος επιτροπής: Ανδρέας Κατσιώτης, Καθηγητής
Μέλος επιτροπής: Παναγιώτης Λουκάκος, Κύριος Ερευνητής FORTH-IESLComplete
Scalar soliton quantization with generic moduli
No full textThis article is distributed under the terms of the Creative Commons
Attribution License (CC-BY 4.0), which permits any use, distribution and reproduction in
any medium, provided the original author(s) and source are credArticle funded by SCOAP3. CP is
a Royal Society Research Fellow and partly supported by the U.S. Department of Energy
under grants DOE-SC0010008, DOE-ARRA-SC0003883 and DOE-DE-SC0007897. ABR
is supported by the Mitchell Family Foundation. We would like to thank the Mitchell
Institute at Texas A&M and the NHETC at Rutgers University respectively for hospitality
during the course of this work. We would also like to acknowledge the Aspen Center
for Physics and NSF grant 1066293 for a stimulating research environment which led to
questions addressed in this paper
Picosecond resonance Raman evidence of the structure of a long-lived electronic excited state of low-spin Fe(III)hemeo
No full textThe structure of the excited state of low-spin ferric heme o has been studied using picosecond time-resolved resonance Raman spectroscopy. The excited state has a lifetime of 10-30 ps, and the heme skeletal vibrations are shifted to lower frequencies relative to those in the electronic ground state. Based on the relatively small frequency shifts, we conclude that the relatively long-lived heme o excited state is not a porphyrin ππ* state. We propose that the heme o excited state is a charge transfer state, in which the heme iron donates an electron to the porphyrin macrocycle
Oxygen-linked equilibrium Cu B-CO species in cytochrome ba 3 oxidase from Thermus thermophilus: implications for an oxygen channel at the Cu B site
No full textWe report the first study of O 2 migration in the putative O 2 channel of cytochrome ba 3 and its effect to the properties of the binuclear heme a 3-Cu B center of cytochrome ba 3 from Thermus thermophilus. The Fourier transform infrared spectra of the ba 3-CO complex demonstrate that in the presence of 60-80 μM O 2, the ν(C-O) of Cu B 1+-C-O at 2053 cm -1 (complex A) shifts to 2045 cm -1 and remains unchanged in H 2O/D 2O exchanges and in the pH 6.5-9.0 range. The frequencies but not the intensities of the C-O stretching modes of heme a 3-CO (complex B), however, remain unchanged. The change in the ν(C-O) of complex A results in an increase of k -2, and thus in a higher affinity of Cu B for exogenous ligands. The time-resolved step-scan Fourier transform infrared difference spectra indicate that the rate of decay of the transient Cu B 1+-CO complex at pH 6.5 is 30.4 s -1 and 28.3 s -1 in the presence of O 2. Similarly, the rebinding to heme a 3 is slightly affected and occurs with k 2 = 26.3 s -1 and 24.6 s -1 in the presence of O 2. These results provide solid evidence that in cytochrome ba 3, the ligand delivery channel is located at the Cu B site, which is the ligand entry to the heme a 3 pocket. We suggest that the properties of the O 2 channel are not limited to facilitating ligand diffusion to the active site but are extended in controlling the dynamics and reactivity of the reactions of ba 3 with O 2 and N
Dioxygen Reduction by Cytochrome Oxidase: A Proton-Transfer Limited Reaction
No full textThe kinetic constraints that are imposed on cytochrome oxidase in its dual function as the terminal oxidant in the respiratory process and as a redox-linked proton pump provide a unique opportunity to investigate the molecular details of biological O2 activation. By using flow/flash techniques, it is possible to visualize individual steps in the O2-binding and reduction process, and results from a number of spectroscopic investigations on the oxidation of reduced cytochrome oxidase by O2 are now available. In this article, we use these results to synthesize a reaction mechanism for O2 activation in the enzyme and to simulate time-concentration profiles for a number of intermediates that have been observed experimentally. Kinetic manifestations of the consequences of coupling exergonic electron transfer to endergonic proton translocation emerge from this analysis. An important conclusion is that, in achieving efficiency in this redox-coupled proton translocation mechanism, rate limitation in dioxygen activation in cytochrome oxidase is transferred to protonation reactions that occur late in the reduction reaction. As a consequence, potentially toxic intermediate oxidation states of dioxygen accumulate to substantial concentration during the reduction reaction, which allows us to detect and characterize these species
Time-resolved resonance Raman detection of intermediates in the reduction of dioxygen by cytochrome oxidase
No full textThesis (Ph. D.)--Michigan State University. Chemical Physics Program, 1990Includes bibliographical references (pages 119-123
Light harvesting and photoprotective states in the marine diatom Fragilariopsis sp.: functional implications of chlorophylls c 1/c 2 in the fucoxanthin-chlorophyll a/c-binding proteins (FCPs)
No full textWe report pH/pD-dependent fluorescence-excitation spectra of the light-harvesting fucoxanthin-chlorophyll a/c-binding proteins (FCPs) of the marine diatom Fragilariopsis sp. There is a reversible 451 to 455 nm Soret transition accompanied by a 588 to 586 nm Qx transition of chlorophylls c 1/c 2 in the pH/pD 4.9-8 range with a pK a = 5.4. The H/D exchangeable site of the 17-acrylate group of chlorophylls c 1/c 2 was characterized and from the pH/pD sensitivity of the Soret and Qy bands we suggest that the chlorophylls c 1/c 2-acrylate-H2O moiety can act as a proton acceptor/donor site. Under high intensity light conditions, the acrylate moiety of chlorophylls c 1/c 2 becomes protonated, resembling that observes under acidic conditions. In the photoprotective state, the absorption of chlorophylls c 1/c 2 is red shifted and resembles that observed in the reversible transition from light-harvesting to energy-quenching state at acidic pH. The induced ΔpH and that created from the high intensity light, is responsible for the red-shifted chlorophylls c 1/c 2 due to the protonation of the acrylate group. We present a model that describes an open and a closed form of the protonated/deprotonated chlorophylls c 1/c 2-acrylate-H2O moiety that controls the proton loading site in the photoprotective and light harvesting state.This work was supported from funds from Cyprus University of Technology
- …
