Electrochemistry of niobium(V) in sulfuric and methanesulfonic acids: formation of the Nb3O2(SO4)6(H2O)(3)(5-) cluster and designed electrochemical generation of "Nb3O2" core clusters by double potential pulse electrolysis

The electrochemical and spectroelectrochemical properties of niobium(V) and the Nb(3)O(2)(SO(4))(6)(H(2)O)(3)(5-) cluster in sulfuric acid and methanesulfonic acid were investigated using cyclic voltammetry, constant potential electrolysis, and spectroelectrochemistry. These chemical systems were suitable to probe the formation of "Nb(3)O(2)" core trinuclear clusters. In 9 M H(2)SO(4) the cluster Nb(3)O(2)(SO(4))(6)(H(2)O)(3)(5-) exhibited a reversible 1-electron reduction peak at E(pc) = -1.30 V vs Hg/Hg(2)SO(4) electrode, as well as a 4-electron irreversible oxidation peak at E(pa) = -0.45 V. Controlled potential reduction at E = -1.40 V produced the green Nb(3.33+) cluster anion Nb(3)O(2)(SO(4))(6)(H(2)O)(3)(6-). In 12 M H(2)SO(4) Nb(V) displayed two reduction peaks at E(pc) = -1.15 V and E(pc) = -1.30 V. It was determined that the first process involves a quasi-reversible 2-electron reduction. After reduction of Nb(V) to Nb(III) the following chemical step involves formation of [Nb(III)](2) dimer, which further reacts with Nb(V) to produce the Nb(3)O(2)(SO(4))(6(H(2)O)(3)(5-) cluster (ECC process). The second reduction peak at E(pc) = -1.30 V corresponds to further 2-electron reduction of Nb(III) to Nb(I). The electrogenerated Nb(I) species also chemically reacts with starting material Nb(V) to produce additional [Nb(III)](2). In 5 M H(2)SO(4), the rate of the second chemical step in the ECC process is relatively slower and reduction of Nb(V) at E = -1.45 V/-1.2 V produces a mixture of Nb(3)O(2)(SO(4))(6)(H(2)O)(3)(5-) and [Nb(III)](2) dimer. [Nb(III)](2) can be selectively oxidized by two 2-electron steps at E = -0.65 V to Nb(V). However, if the oxidation is performed at E = -0.86 V, the product is Nb(3)O(2)(SO(4))(6)(H(2)O)(3)(5-). A double potential pulse electrolysis waveform was developed to direct the reduction of Nb(V) toward selective formation of the Nb(3)O(2)(SO(4))(6)(H(2)O)(3)(5-) cluster. Proper application of dc-voltage pulses alternating between E(1) = -1.45 V and E(2) = -0.86 V yields only the target trinuclear cluster. Analogous double potential pulse electrolysis of Nb(V) in methanesulfonic acid generates the "Nb(3)O(2)" core cluster Nb(3)O(2)(CH(3)SO(3))(6)(H(2)O)(3)(+).     

Use of an internal control for matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis of bacteria

A method to aid in the analysis of bacterial samples of unknown concentration by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry is demonstrated. It is shown that in MALDI analysis of bacteria, the intensities of resulting peaks in spectra are sensitive to the microbial concentration. At the high and low ends of the concentration range, no signal can be obtained, leaving very concentrated or very dilute samples indistinguishable. The addition of cytochrome c as an internal control allows the differentiation of these concentrated and dilute samples. The presence of the internal control causes only a 20% to 30% decrease in signal intensity when the bacterial concentration is optimum. However, the signal quality is improved when the internal control is added to some low concentrations of bacteria.     

EXCITATION ENERGY MIGRATION IN PHYCOBILISOMES: COMPARISON OF EXPERIMENTAL RESULTS AND THEORETICAL PREDICTIONS

Abstract— Quantum yield and fluorescence polarization determinations on phycobilisomes and their constituent phycobiliproteins show that phycobilisomes are energetically effective macromolecular structures. Energy migration within the phycobilisome to allophycocyanin, the longest wavelength absorbing and emitting phycobiliprotein, was indicated by the predominant allophycocyanin fluorescence emission which was independent of the phycobiliprotein being excited. The high efficiency of the energy migration inside the phycobilisome was reflected by the low polarized fluorescence. Excitation of phycobilisomes in the region of major absorption (500–650 nm) resulted in degrees of fluorescence polarization between +0.02 and –0.02, whereas in isolated phycobiliproteins the values were 2 to 12 times greater. Furthermore, 94–98° of the excitation energy of phycoerythrin was transferred to phycocyanin and allophycocyanin as determined from comparisons of fluorescence spectra of intact and dissociated phycobilisomes. The fluorescence quantum yields of phycobilisomes were about 0.60–0.68, very similar to that of pure allophycocyanin in solution (0.68). Phycobilisomes isolated from Fremyella diplosiphon and Nostoc sp. (blue-gree algae) have respective quantum yields of 0.68 and 0. 65, and those isolated from Porphyridium cruentum (red alga), about 0.60. In Fremyella diplosiphon and Nostoc sp., which showed a striking adaptation to different wavelengths, the phycobilisome quantum yields only varied from 0.68 to 0.67 and from 0.65 to 0. 60, respectively. The mean transfer time, calculated on the basis of experimental results, was about 280 ± 40 ps for transfer of excitation from the phycoerythrin to the phycocyanin layer in phycobilisomes. This time corresponds to the mean number of jumps, about 28, of the excitation in the phycoerythrin layer before it is captured by phycocyanin. These values are in reasonable agreement with the values of 250 ± 30 ps and 25 jumps, calculated on the basis of a phycobilisome model (of Porphyridium cruentum) and Pearlstein's theory of energy migration devised for a three-dimensional photosynthetic unit. It was also shown that Paillotin's theory of energy migration predicts similar values for mean transfer time and mean number of jumps, if one assumes that phycocyanin is a perfect sink for phycoerythrin excitation.     

SPECTRAL PROPERTIES OF PHYCOBILISOMES AND PHYCOBILIPROTEINS FROM THE BLUE-GREEN ALGA-NOSTOC SP.*

Abstract— An improved method for phycobilisome isolation from a blue-green alga Nostoc sp. was developed using 1% Triton X-100. The phycobilisome preparations showed little fragmentation and had structures similar in size to those observed in thin sections of the organism. Phycobiliproteins isolated from phycobilisomes and examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis, had subunits with the following molecular weights: phycoerythrin (PE), 20,000 and 16,900; phycocyanin (PC), 14,700 and 16,300; and allophycocyanin (APC), 14,000. Isoelectric focusing of each phycobiliprotein resulted in major bands isoelectric at the following pH values: PE, 4.43, 4.45; PC 4.32; APC, 4.38. Absorption spectra at -196°c showed maxima at 551 and 566 nm for PE; 598 and 631 nm for PC; and 590, 600, 629 and 650 nm for APC. Concentrated vs dilute difference spectra of phycobiliproteins showed increased absorption at 574 nm (PE), 630 nm (PC) and 651 nm (APC) suggesting that spectral changes resulted from aggregation. Fluorescence analysis of each phycobiliprotein and of intact phycobilisome preparations showed that energy absorbed by phycoerythrin is transferred to allophycocyanin, possibly by a resonance transfer mechanism. These observations support a model where allophycocyanin forms the base of the phycobilisome which is attached to the photosynthetic membrane. The next layer is assumed to be phycocyanin, which in turn is followed by a phycoerythrin layer that is the outermost layer (on the stroma side) of the phycobilisome.     

ALLOPHYCOCYANIN FORMS ISOLATED FROM NOSTOC SP. PHYCOBILISOMES*

Abstract— Allophycocyanin from dissociated phycobilisomes of Nostoc sp. occurs in three spectrally identifiable forms that fractionate on calcium phosphate adsorption chromatography as: allophycocyanin (APC) I (15–20%), APC II (4&50%), and APC III (30–40%). APC I has a single absorption maximum at 654 nm, and a fluorescence emission peak at 678 nm. The absorption peaks of APC II and III are both at 650 nm, but the relative absorbance at 620/650 nm of APC III is less than that of APC II. The emission of both is maximum at 660 nm. On zone sedimentation in sucrose, their S_20,w values of 6.0 ± 0.1 (APC I), 5.0 ± 0.1 (APC II), and 5.3 ± 0.2 (APC III) were comparable to the order of their elution from Sephadex G-200. On SDS acrylamide gel electrophoresis two subunits were resolved with apparent molecular weights of 16,900 and 18,400 daltons. When stained by Coomassie blue, they were present in a ratio of 1α:1β in APC II and III, and a probable ratio of 2a:3β in APC I. The larger size of APC I may be accounted for by additional β subunits, by the presence of an additional polypeptide of 35,000 daltons, or both. Over several days, bleaching as noted by a decrease in absorbance at 650 nm, occurred in all three forms; in addition, the more pronounced bleaching at 650 nm, relative to 620 nm, results in APC III becoming spectrally identical to APC II. A trace of a fourth pigment, probably comparable to allophycocyanin-B, was occasionally detected. The results suggest that several in vitro APC forms (sharing similar subunits) arise upon phycobilisome dissociation, and that APC I is the form most closely related to the final fluorescence emitter of intact phycobilisomes. In this form it probably serves as the bridging pigment in energy transfer from the phycobilisomes to chlorophyll.     

POLARIZED PHOTO ACOUSTIC SPECTRA OF PHYCOBILISOMES IN POLYVINYL ALCOHOL FILMS

Abstract— Phycobilisomes from Porphyridium cruentum , suspended in polyvinyl alcohol were found to be highly stable, and had normal absorption and fluorescence spectra. Intact phycobilisomes had a major emission peak at 680 nm, whereas upon partial dissociating the major emission was at 580 nm. Incorporation of phycobilisomes in thin polyvinyl alcohol films facilitated examination by photoacoustic spectroscopy. The photoacoustic spectra had a broad absorption maximum at 545–565 nm (phycoerythrin), which resolved as two peaks (545 and 563 nm) in absorption spectra. Stretching of films resulted in apparent chromophore reorientation in partially dissociated, but not in intact phycobilisomes. Only in dissociated phycobilisomes was observed a differential chromophore orientation at 685 nm by polarized fluorescence, which is attributed to a change in orientation of the terminal phycobilisome pigment relative to phycoerythrin.     

Enzymatic methods for glyco(diversification/randomization) of drugs and small molecules

Glyco (randomization/diversification) is a term that encompasses strategies to diversify a core drug scaffold via enzymatic glycosylation to provide sets of analogs wherein the sole diversity element is a carbohydrate. This review covers the influence of glycosylation upon various drug properties, the classes of glycosyl-conjugating enzymes amenable to glyco(randomization/diversification) schemes, approaches to the synthesis of required substrates and specific examples of glycorandomized libraries utilizing both wild-type and engineered enzymes.