Fluorescence-based imaging of cellular defect in lysinuric protein intolerance (LPI)

Fluoresenssiperusteiset kuvantamismenetelmät lysinurisen proteiini-intoleranssin (LPI) soluhäiriön tutkimuksessa Lysinurinen proteiini-intoleranssi on suomalaiseen tautiperintöön kuuluva autosomaalisesti peit¬tyvästi periytyvä sairaus, jonka aiheuttaa kationisten aminohappojen kuljetushäiriö munuaisten ja ohutsuolen epiteelisolujen basolateraalikalvolla. Aminohappojen kuljetushäiriö johtaa moniin oirei¬siin, kuten kasvuhäiriöön, osteoporoosiin, immuunijärjestelmän häiriöihin, oksenteluun ja runsaspro¬teiinisen ravinnon nauttimisen jälkeiseen hyperammonemiaan. LPI-geeni SLC7A7 (solute carrier family 7 member 7) koodaa y+LAT1 proteiinia, joka on basolateraali¬nen kationisten ja neutraalien aminohappojen kuljettimen kevyt ketju, joka muodostaa heterodimee¬rin raskaan alayksikön 4F2hc:n kanssa. Tällä hetkellä SLC7A7-geenistä tunnetaan yli 50 LPI:n aiheut¬tavaa mutaatiota. Tässä tutkimuksessa erityyppisiä y+LAT1:n LPI-mutaatiota sekä yhdeksän C-terminaalista polypep¬tidiä lyhentävää deleetiota kuvannettiin nisäkässoluissa y+LAT1:n GFP (green fluorescent protein) -fuusioproteiineina. Tulokset vahvistivat muissa soluissa tehdyt havainnot siitä, että 4F2hc on edel¬lytyksenä y+LAT1:n solukalvokuljetukselle, G54V-pistemutantti sijaitsee solukalvolla samoin kuin vil¬lityyppinen proteiini, mutta lukukehystä muuttavia ja proteiinia lyhentäviä mutantteja ei kuljeteta solukalvoon. Lisäksi havaittiin, että poikkeuksena tästä säännöstä ovat y+LAT1-deleetioproteiinit, joista puuttui korkeintaan 50 C-terminaalista aminohappoa. Nämä lyhentyneet kuljettimet sijaitsevat solukalvolla kuten villityyppiset ja LPI-pistemutanttiproteiinit. Dimerisaation osuutta kuljetushäiriön synnyssä tutkittiin käyttämällä fluorescence resonance energy transfer (FRET) menetelmää. Heterodimeerin alayksiköistä kloonattiin ECFP (cyan) ja EYFP (yellow) fuusioproteiinit, joita ilmennettiin nisäkässoluissa, ja FRET mitattiin virtaussytometri-FRET -menetel¬mällä (FACS-FRET). Tutkimuksissa kaikkien mutanttien havaittiin dimerisoituvan yhtä tehokkaasti. Kul¬jetushäiriön syynä ei siten ole alayksiköiden dimerisaation estyminen mutaation seurauksena. Tutkimuksessa havaittiin, että kaikki mutantti-y+LAT1-transfektiot tuottavat vähemmän transfektoi¬tuneita soluja kuin villityyppisen y+LAT1:n transfektiot. Solupopulaatioissa, joihin oli tranfektoitu lu¬kukehystä muuttava tai stop-kodonin tuottava mutaatio havaittiin suurempi kuolleisuus kuin saman näytteen transfektoitumattomissa soluissa, kun taas villityyppistä tai G54V-pistemutanttia tuottavas¬sa solupopulaatiossa oli pienempi kuolleisuus kuin saman näytteen fuusioproteiinia ilmentämättö¬missä soluissa. Tulos osoittaa mutanttiproteiinien erilaiset vaikutukset niitä ilmentäviin soluihin, joko suoraan y+LAT1:n tai 4F2hc:n kautta aiheutuneina. LPIFin SLC7A7 lähetti-RNA:n määrä ei merkittävästi poikennut villityyppisen määrästä fibroblasteissa ja lymfoblasteissa. SLC7A7:n promoottorianalyysissä oli osoitettavissa säätelyalueita geenin 5’ ei-koo¬daavalla alueella sekä ensimmäisten kahden intronin alueella. LPI-taudin tautimekanismin kannalta keskeisin tekijä on kuitenkin aminohappokuljetuksen häiriö, jonka vaikutuksesta näistä aminohapoista riippuvaiset prosessit elimistössä eivät toimi normaalisti. Havaittu virheellinen y+LAT1/4F2hc kuljetuskompleksin sijainti edellyttää lisätutkimuksia sen mahdol¬lisen kliinisen merkityksen selvittämiseksi.

Chlorophyll a fluorescence in rice plants exposed of herbicides of group imidazolinone

The objective of this work was to investigate the injuries caused to the photosynthetic apparatus of three types of rice exposed to application of imidazolinone group herbicides. Two experiments were conducted using herbicides Imazethapyr+imazapic and Imazapyr+imazapic, in a split-plot experimental design, and a 3 x 3 factorial, with six replications. The first factor (A) consisted of the herbicide rates 0, 100 e 200 g ha-1 of Imazethapyr+imazapic and 0, 140 e 280 g ha-1 of Imazapyr+imazapic; factor B consisted of type of rice (cv. Puitá Inta CL, sensitive red rice ecotype and red rice ecotype with suspected herbicide tolerance to Imidazolinone). Chlorophyll a fluorescence parameters were evaluated in plants at 30 days after herbicide application, using a portable fluorometer (HandyPEA, Hanstech). The photosynthetic metabolism of cv. Puitá Inta CL was found to tolerate commercial dosages of both herbicides. High sensitivity to the herbicides was observed for the sensitive red rice ecotype, while the photosynthetic apparatus of red rice ecotype with suspected herbicide tolerance showed high tolerance to both herbicides applied at rates higher than the commercial rate. The application of chemical herbicides of the imidazolinone group on rice plants causes changes in the photosynthetic metabolism of plants, detected by evaluating the emission of transient chlorophyll a fluorescence. This method can be useful in helping detect resistance and/or tolerance of red rice plants to herbicides of the imidazolinone group.

Chlorophyll fluorescence in guanandi tree (Calophyllum brasiliense) after herbicide application

Chlorophyll fluorescence is currently used as a rapid diagnostic and nondestructive method to detect and quantify damage on the photosynthetic apparatus of leaves on weeds, crops and ornamental/coniferous trees in response to both environmental stress and herbicides. This study aimed to evaluate chlorophyll fluorescence in guanandi plants (Calophyllum brasiliense) after application of different postemergence herbicides. The experiment was performed in a completely randomized design, with six treatments (control, bentazon, sulfentrazone, isoxaflutole, atrazine and glyphosate) and five replications. The herbicide treatments were applied with a stationary sprayer, and electron transport rate (ETR) was subsequently analyzed with OS5p Multi-Mode Chlorophyll Fluorometer. In the monitored period, guanandi plants subjected to atrazine showed higher sensitivity to chlorophyll fluorescence than the other treatments. Although bentazon is a photosystem II inhibitor, it showed no major changes in electron transport for the studied species and in the monitored period. In summary, ETR is a good parameter to evaluate the effect of some herbicides on Calophyllum brasiliense plants.

Chlorophyll fluorescence parameters in populations of two legume trees: Stryphnodendron adstringens (Mart.) Coville (Mimosoideae) and Cassia ferruginea (Schrad.) Schrad. ex DC. (Caesalpinoideae)

The aim of this study was to investigate the photosynthetic performance in populations of two legume tree species, Stryphnodendron adstringens (Mimosoideae), typical from Cerrado, and Cassia ferruginea (Caesalpinoideae) from the Atlantic Rain Forest. The photosynthetic traits were assessed by measures of chlorophyll fluorescence in progenies of naturally pollinated plants from three populations of S. adstringens and a population of C. ferruginea. Plants of S. adstringens growing under similar conditions of C. ferruginea plants demanded higher light values for photosynthesis saturation, 600 µmol.m-2.s-1 and 350 µmol.m-2.s-1 respectively, and showed higher intrinsic photosynthetic efficiency of photosystem II, Fv/Fm of 0.814 versus 0.783 in C. ferruginea. The highest values of Fv/Fm observed in S. adstringens can explain the highest electron transport rates (ETR) obtained for this species. No significant differences were found among progenies from different C. ferruginea trees nor among populations of S. adstringens, and only in few cases, variation among progenies within populations were found for S. adstringens plants. The fact that fluorescence parameters distinguished species but not populations or most of progenies may be related to low intraspecific genetic variation of these chlorophyll fluorescence traits or due to lack of expression on genetic differences in plants under no stressful conditions.

Interphase cytogenetics using fluorescence in situ hybridization: an overview of its application to diffuse and solid tissue

Interphase cytogenetics, utilizing fluorescence in situ hybridization (FISH) techniques, has been successfully applied to diffuse and solid tissue specimens. Most studies have been performed on isolated cells, such as blood or bone marrow cells; a few have been performed on cells from body fluids, such as amniotic fluid, urine, sperm, and sputum. Mechanically or chemically disaggregated cells from solid tissues have also been used as single cell suspensions for FISH. Additionally, intact organized tissue samples represented by touch preparations or thin tissue sections have been used, especially in cancer studies. Advantages and pitfalls of application of FISH methodology to each type of specimen and some significant biological findings achieved are illustrated in this overview.

JAK/STAT-deficient cell lines

Mutant cell lines B3 and B10, which are unresponsive to both interferon (IFN)-alpha and IFN-gamma, and line B9, which does not respond to IFN-gamma stimulation, are described. The mutants were submitted to fluorescence-activated cell sorting (FACS) from a cellular pool, which was obtained from the parental cell line 2C4 after several rounds of mutagenesis. The unresponsiveness to IFN stimulation was observed both in terms of expression of cell surface markers (CD2, class I and II HLAs) and mRNA expression of IFN-stimulated genes (2'-5' oligoadenylate synthetase (OAS), 9-27, and guanylate binding protein (GBP)). Genetic crossing of B3, B9 and B10 with U3 (STAT1-), gamma2a (JAK2-) and U4 (JAK1-) mutants, respectively, did not restore IFN responsiveness to the hybrid cell lines. However, when these cell lines were crossed with the same mutants, but using the pairwise crosses B3 x U4, B9 x U3 and B10 x U3, the cell hybrids recovered full IFN responsiveness. The present genetic experiments permitted us to assign the mutant cell lines B3, B9 and B10 to the U3, gamma2 and U4 complementation groups, respectively. These conclusions were supported by the analysis of IFN-stimulated genes in the mutants.

A simple HPLC-fluorescence method for the measurement of R,S-sotalol in the plasma of patients with life-threatening cardiac arrhythmias

R,S-sotalol, a ß-blocker drug with class III antiarrhythmic properties, is prescribed to patients with ventricular, atrial and supraventricular arrhythmias. A simple and sensitive method based on HPLC-fluorescence is described for the quantification of R,S-sotalol racemate in 500 µl of plasma. R,S-sotalol and its internal standard (atenolol) were eluted after 5.9 and 8.5 min, respectively, from a 4-micron C18 reverse-phase column using a mobile phase consisting of 80 mM KH2PO4, pH 4.6, and acetonitrile (95:5, v/v) at a flow rate of 0.5 ml/min with detection at lex = 235 nm and lem = 310 nm, respectively. This method, validated on the basis of R,S-sotalol measurements in spiked blank plasma, presented 20 ng/ml sensitivity, 20-10,000 ng/ml linearity, and 2.9 and 4.8% intra- and interassay precision, respectively. Plasma sotalol concentrations were determined by applying this method to investigate five high-risk patients with atrial fibrillation admitted to the Emergency Service of the Medical School Hospital, who received sotalol, 160 mg po, as loading dose. Blood samples were collected from a peripheral vein at zero, 0.5, 1.0, 1.5, 2.0, 3.0, 4.0, 6.0, 8.0, 12.0 and 24.0 h after drug administration. A two-compartment open model was applied. Data obtained, expressed as mean, were: CMAX = 1230 ng/ml, TMAX = 1.8 h, AUCT = 10645 ng h-1 ml-1, Kab = 1.23 h-1, a = 0.95 h-1, ß = 0.09 h-1, t(1/2)ß = 7.8 h, ClT/F = 3.94 ml min-1 kg-1, and Vd/F = 2.53 l/kg. A good systemic availability and a fast absorption were obtained. Drug distribution was reduced to the same extent in terms of total body clearance when patients and healthy volunteers were compared, and consequently elimination half-life remained unchanged. Thus, the method described in the present study is useful for therapeutic drug monitoring purposes, pharmacokinetic investigation and pharmacokinetic-pharmacodynamic sotalol studies in patients with tachyarrhythmias.

Studies on ATP-diphosphohydrolase nucleotide-binding sites by intrinsic fluorescence

Potato apyrase, a soluble ATP-diphosphohydrolase, was purified to homogeneity from several clonal varieties of Solanum tuberosum. Depending on the source of the enzyme, differences in kinetic and physicochemical properties have been described, which cannot be explained by the amino acid residues present in the active site. In order to understand the different kinetic behavior of the Pimpernel (ATPase/ADPase = 10) and Desirée (ATPase/ADPase = 1) isoenzymes, the nucleotide-binding site of these apyrases was explored using the intrinsic fluorescence of tryptophan. The intrinsic fluorescence of the two apyrases was slightly different. The maximum emission wavelengths of the Desirée and Pimpernel enzymes were 336 and 340 nm, respectively, suggesting small differences in the microenvironment of Trp residues. The Pimpernel enzyme emitted more fluorescence than the Desirée apyrase at the same concentration although both enzymes have the same number of Trp residues. The binding of the nonhydrolyzable substrate analogs decreased the fluorescence emission of both apyrases, indicating the presence of conformational changes in the neighborhood of Trp residues. Experiments with quenchers of different polarities, such as acrylamide, Cs+ and I- indicated the existence of differences in the nucleotide-binding site, as further shown by quenching experiments in the presence of nonhydrolyzable substrate analogs. Differences in the nucleotide-binding site may explain, at least in part, the kinetic differences of the Pimpernel and Desirée isoapyrases.

Determination of macromolecular exchange and PO2 in the microcirculation: a simple system for in vivo fluorescence and phosphorescence videomicroscopy

We have developed a system with two epi-illumination sources, a DC-regulated lamp for transillumination and mechanical switches for rapid shift of illumination and detection of defined areas (250-750 µm²) by fluorescence and phosphorescence videomicroscopy. The system permits investigation of standard microvascular parameters, vascular permeability as well as intra- and extravascular PO2 by phosphorescence quenching of Pd-meso-tetra (4-carboxyphenyl) porphine (PORPH). A Pechan prism was used to position a defined region over the photomultiplier and TV camera. In order to validate the system for in vivo use, in vitro tests were performed with probes at concentrations that can be found in microvascular studies. Extensive in vitro evaluations were performed by filling glass capillaries with solutions of various concentrations of FITC-dextran (diluted in blood and in saline) mixed with different amounts of PORPH. Fluorescence intensity and phosphorescence decay were determined for each mixture. FITC-dextran solutions without PORPH and PORPH solutions without FITC-dextran were used as references. Phosphorescence decay curves were relatively unaffected by the presence of FITC-dextran at all concentrations tested (0.1 µg/ml to 5 mg/ml). Likewise, fluorescence determinations were performed in the presence of PORPH (0.05 to 0.5 mg/ml). The system was successfully used to study macromolecular extravasation and PO2 in the rat mesentery circulation under controlled conditions and during ischemia-reperfusion.

Immunophenotype of normal and leukemic bone marrow B-precursors in a Brazilian population. A comparative analysis by quantitative fluorescence cytometry

The distinction between normal and leukemic bone marrow (BM) B-precursors is essential for the diagnosis and treatment monitoring of acute lymphoblastic leukemia (ALL). In order to evaluate the potential use of quantitative fluorescence cytometry (QFC) for this distinction, we studied 21 normal individuals and 40 patients with CD10+ ALL. We characterized the age-related changes of the CD10, CD19, TdT, CD34 and CD79a densities in normal and leukemic BM. Compared to normal adults, the B-precursors from normal children expressed significantly lower values of CD34-specific antibody binding capacity (SABC) (median value of 86.6 vs 160.2 arbitrary units (a.u.) in children and adults, respectively). No significant age-related difference was observed in the expression of the other markers in the normal BM, or in any of the markers in the leukemic BM. Based on the literature, we set the cut-off value for the normal CD10 expression at 45 x 10³ a.u. for both age groups. For the remaining markers we established the cut-off values based on the minimum-maximum values in the normal BM in each age group. The expression of CD10 was higher than the cut-off in 30 ALL cases and in 18 of them there was a concomitant aberrant expression of other markers. In 9 of the 10 CD10+ ALL with normal CD10 SABC values, the expression of at least one other marker was aberrant. In conclusion, the distinction between normal and leukemic cells by QFC was possible in 38/40 CD10+ ALL cases.

Quenching of the intrinsic fluorescence of bovine serum albumin by chlorpromazine and hemin

The binding of chlorpromazine (CPZ) and hemin to bovine serum albumin was studied by the fluorescence quenching technique. CPZ is a widely used anti-psychotic drug that interacts with blood components, influences bioavailability, and affects function of several biomolecules. Hemin is an important ferric residue of hemoglobin that binds within the hydrophobic region of albumin with high specificity. Quenching of the intrinsic fluorescence of bovine serum albumin (BSA) was observed by selectively exciting tryptophan residues at 290 nm. Emission spectra were recorded in the range from 300 to 450 nm for each quencher addition. Stern-Volmer graphs were plotted, and the quenching constant estimated for BSA solution titrated with hemin at 25ºC was 1.44 (± 0.05) x 10(5) M-1. Results showed that bovine albumin tryptophans are not equally accessible to CPZ, in agreement with the idea that polar or charged quenchers have more affinity for amino acid residues on the outer wall of the protein. Hemin added to albumin solution at a molar ratio of 1:1 quenched about 25% of their fluorescence. The quenching effect of CPZ on albumin-hemin solution was stronger than on pure BSA. This increase can be the result of combined conformational changes in the structure of albumin caused firstly by hemin and then by CPZ. Our results suggest that the primary binding site for hemin on bovine albumin may be located asymmetrically between the two tryptophans along the sequence formed by subdomains IB and IIA, closer to tryptophan residue 212.

Use of chlrophyll fluorescence sorting to improve soybean seed quality

The occurrence of green seeded soybeans [Glycine max (L.) Merrill] is a problem closely related to unfavorable climatic conditions, mainly drought, that occurs during the final stages of seed maturation. This problem causes serious losses to soybean seed quality in Brazil. In these seeds, chlorophyll is not properly degraded during maturation, drastically reducing seed quality. Using the chlorophyll fluorescence technique, it is possible to remove green seeds from the seed lot, improving seed quality in several species in which the occurrence of green seeds is also a problem. The objective of this research was to study the use of the chlorophyll fluorescence technique in sorting green seeds from soybean seed samples and its effects on quality. Five seed samples of soybean, cultivar TMG 113 RR, with 0%, 5%, 10%, 15%, and 20% of green seeds were used in this study. Seeds from each sample were sorted into two fractions based on the chlorophyll fluorescence signals and then compared to the control (non-sorted seeds). The sorting process showed great differences between the low and high chlorophyll fluorescence fractions. It was concluded that: green seeds of soybeans present high chlorophyll fluorescence and that this characteristic affects the quality of the seeds; it is possible to improve the quality of soybean seed by removing green seeds using the chlorophyll fluorescence sorting technique.

Aikaerotteisesti fluoresenssia mittaavan reaaliaikaisen PCR-laitteen kehitys

Kvantitatiivinen reaaliaikainen polymeraasiketjureaktio (engl. polymerase chain reaction, PCR) on osoittautunut käyttäjäystävällisimmäksi menetelmäksi nukleiinihapposekvenssien kvantitoimisessa. Tätä menetelmää voidaan herkistää pienempien DNA-pitoisuuksien havaitsemiseen käyttämällä hyväksi aikaerotteista fluorometriaa (engl. time-resolved fluorometry, TRF) ja luminoivia lantanidileimoja, joiden fluoresenssin pitkän eliniän ansiosta emission mittaus voidaan suorittaa vasta hetki virittävän valopulssin jälkeen, jolloin lyhytikäinen taustasäteily ehtii sammua. Tuloksena saadaan korkea signaali-taustasuhde. Tämän diplomityön tarkoituksena oli rakentaa TRF:än pystyvä reaaliaikainen PCR-laite, sillä tällaista laitetta ei ole markkinoilla tarjolla. Laite rakennettiin kehittämällä lämpökierrätin ja yhdistämällä se valmiiseen TRF:än kykenevään mittapäähän. Mittapään ja lämpökierrättimen hallitsemiseksi kehitettiin myös tietokoneohjelma. Valon tuottamiseksi ja mittaamiseksi haluttiin käyttää edullisia komponentteja, joten työssä käytettiin valmiin mittapään optiikkaa, jossa viritys tapahtuu hohtodiodilla (engl. light-emitting diode, LED) ja lantanidileiman emission mittaus fotodiodilla (engl. photodiode, PD) tai valomonistinputkella (engl. photomultiplier tube, PMT). Myös mittapään suorituskykyä tutkittiin. Työtä varten kehitettiin lämpökierrätin, joka koostui Peltier-elementillä lämmitettävästä PCR-putkitelineestä ja lämpökannesta. Mittalaitteen suorituskyvyn tutkimiseen käytettiin kelaattikomplementaatioon perustuvaa PCR-tuotteen havaitsemismenetelmää. Kelaattikomplementaatio perustuu kahteen erilliseen oligonukleotidimolekyyliin, joista toiseen on sidottu lantanidi-ioni ja toiseen valoa absorboiva ligandirakenne, jotka yhdessä muodostavat fluoresoivan kokonaisuuden. Kehitetyn lämpökierrättimen todettiin olevan tarpeeksi tarkka sekä tehokas ja sen lämmitys- ja jäähdytysnopeuden maksimeiksi saatiin 2,6 °C/sekunti. Detektorina käytetyn PD:n ei todettu olevan tarpeeksi herkkä emission havainnoimiseksi ja se korvattiin laitteessa PMT:llä. Käytetyllä PCR-määrityksellä kynnyssykleiksi (engl. threshold cycle, Ct) sekä kehitetylle että referenssilaitteelle saatiin 28,4 käyttämällä samaa 100 000 kopion DNA:n aloitusmäärää. Työssä osoitettiin, että on mahdollista kehittää edullisia komponentteja käyttävä, TRF:än pystyvä, reaaliaikainen PCR-laite, joka kykenee vastaavaan Ct-arvoon kuin vertailulaite. PD:n herkkyys ei kuitenkaan riittänyt. Tulokset olivat lupaavia, sillä LED- ja PD-teknologiat kehittyvät ja markkinoille on tullut myös muita komponentteja, joiden avulla on tulevaisuudessa mahdollista kehittää vielä herkempi laite.

Investigation of single tryptophan proteins encapsulated in TEOS-derived sol-gel matrices by fluorescence spectroscopy /

In the work reported here, optically clear, ultrathin TEOS derived sol-gel slides which were suitable for studies of tryptophan (Trp) fluorescence from entrapped proteins were prepared by the sol-gel technique and characterized. The monitoring of intrinsic protein fluorescence provided information about the structure and environment of the entrapped protein, and about the kinetics of the interaction between the entrapped protein and extemal reagents. Initial studies concentrated on the single Trp protein monellin which was entrapped into the sol-gel matrices. Two types of sol-gel slides, termed "wet aged", in which the gels were aged in buffer and "dry-aged", in which the gels were aged in air , were studied in order to compare the effect of the sol-gel matrix on the structure of the protein at different aging stages. Fluorescence results suggested that the mobility of solvent inside the slides was substantially reduced. The interaction of the entrapped protein with both neutral and charged species was examined and indicated response times on the order of minutes. In the case of the neutral species the kinetics were diffusion limited in solution, but were best described by a sum of first order rate constants when the reactions occurred in the glass matrix. For charged species, interactions between the analytes and the negatively charged glass matrix caused the reaction kinetics to become complex, with the overall reaction rate depending on both the type of aging and the charge on the analyte. The stability and conformational flexibility of the entrapped monellin were also studied. These studies indicated that the encapsulation of monellin into dry-aged monoliths caused the thermal unfolding transition to broaden and shift upward by 14°C, and causedthe long-term stability to improve by 12-fold (compared to solution). Chemical stability studies also showed a broader transition for the unfolding of the protein in dry-aged monoliths, and suggested that the protein was present in a distribution of environments. Results indicated that the entrapped proteins had a smaller range of conformational motions compared to proteins in solution, and that entrapped proteins were not able to unfold completely. The restriction of conformational motion, along with the increased structural order of the internal environment of the gels, likely resulted in the improvements in themial and long-term stability that were observed. A second protein which was also studied in this work is the metal binding protein rat oncomodulin. Initially, the unfolding behavior of this protein in aqueous solution was examined. Several single tryptophan mutants of the metal-binding protein rat oncomodulin (OM) were examined; F102W, Y57W, Y65W and the engineered protein CDOM33 which had all 12 residues of the CD loop replaced with a higher affinity binding loop. Both the thermal and the chemical stability were improved upon binding of metal ions with the order apo < Ca^^ < Tb^"^. During thermal denaturation, the transition midpoints (Tun) of Y65W appeared to be the lowest, followed by Y57W and F102W. The placement of the Trp residue in the F-helix in F102W apparently made the protein slightly more thermostable, although the fluorescence response was readily affected by chemical denaturants, which probably acted through the disruption of hydrogen bonds at the Cterminal end of the F-helix. Under both thermal and chemical denaturation, the engineered protein showed the highest stability. This indicated that increasing the number of metal ligating oxygens in the binding site, either by using a metal ion with a higher coordinatenumber (i.e. Tb^*) which binds more carboxylate ligands, or by providing more ligating groups, as in the CDOM33 replacement, produces notable improvements in protein stability. Y57W and CE)OM33 OM were chosen for further studies when encapsulated into sol-gel derived matrices. The kinetics of interaction of terbium with the entrapped proteins, the ability of the entrapped protein to binding terbium, as well as thermal stability of these two entrapped protein were compared with different levels of Ca^"*^ present in the matrix and in solution. Results suggested that for both of the proteins, the response time and the ability to bind terbium could be adjusted by adding excess calcium to the matrix before gelation. However, the less stable protein Y57W only retained at most 45% of its binding ability in solution while the more stable protein CDOM33 was able to retain 100% binding ability. Themially induced denaturation also suggested that CDOM33 showed similar stability to the protein in solution while Y57W was destabilized. All these results suggested that "hard" proteins (i.e. very stable) can easily survive the sol-gel encapsulation process, but "soft" proteins with lower thermodynamic stability may not be able to withstand the sol-gel process. However, it is possible to control many parameters in order to successfully entrap biological molecules into the sol-gel matrices with maxunum retention of activity.

Fluorescence spectra and ab initio theoretical studies of HCOOH /

A fluorescence excitation spectrum of formic acid monomer (HCOOH) , has been recorded in the 278-246 nm region and has been attributed to an n >7r* electron promotion in the anti conformer. The S^< S^ electronic origins of the HCOOH/HCOOD/DCOOH/DCOOD isotopomers were assigned to weak bands observed at 37431.5/37461.5/37445.5/37479.3 cm'''. From a band contour analysis of the 0°^ band of HCOOH, the rotational constants for the excited state were estimated: A'=1.8619, B'=0.4073, and C'=0.3730 cm'\ Four vibrational modes, 1/3(0=0), j/^(0-C=0) , J/g(C-H^^^) and i/,(0-H^yJ were observed in the spectrum. The activity of the antisymmetric aldehyde wagging and hydroxyl torsional modes in forming progressions is central to the analysis, leading to the conclusion that the two hydrogens are distorted from the molecular plane, 0-C=0, in the upper S. state. Ab initio calculations were performed at the 6-3 IG* SCF level using the Gaussian 86 system of programs to aid in the vibrational assignments. The computations show that the potential surface which describes the low frequency OH torsion (twisting motion) and the CH wagging (molecular inversion) motions is complex in the S^ excited electronic state. The OH and CH bonds were calculated to be twisted with respect to the 0-C=0 molecular frame by 63.66 and 4 5.76 degrees, respectively. The calculations predicted the existence of the second (syn) rotamer which is 338 cm'^ above the equilibrium configuration with OH and CH angles displaced from the plane by 47.91 and 41.32 degrees.