Wyniki wyszukiwania - Biblioteka Nauki

1

Fluorescence quenching study of tryptophan residues in fungal lipase from Rhizomucor miehei

100%

Stobiecka A.

Zeszyty Naukowe. Chemia Spożywcza i Biotechnologia / Politechnika Łódzka

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1998

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tom Z. 60

53-63

EN

The iodide-quenching of a four-tryptophan-containing lipase from Rhizomucor miehei has been investigated The results of steady-state fluorescence quenching experiments at various excitation wavelengths have shown the presence of two classes of lipase emission. One of the component, exposed to solvent has an effective Stern-Volmer quenching constant equal to 8.00 ± 0.43 M-1 at λex = 290 nm and 11.90 ± 0.49 M-1 at λex = 300 nm, respectively. The second component was unquenchable by iodide. The fluorescence emission spectra of lipase have been separated into components using a modified Stern-Volmer equation. Obtained results demonstrated that the spectral shifts upon red-edge excitation have been observed only for accessible tryptophans. The fluorescence peak of exposed residues within protein when illuminated at the wavelength range from 290 nm to 300 nm was shifted from 344 ± 1 nm to 354 ± 1 nm. Contrary, the maximum of fluorescence emission of inaccessible tryptophans was equal to 338 ± 1 nm, irrespective to the excitation wavelength used.

PL

W niniejszym artykule przedstawione zostały wyniki wstępnych pomiarów fluorescencyjnych zewnątrzkomórkowej lipazy pleśniowej z Rhizomucor miehei (Rml). Metodami fluorescencji stacjonarnej badano wygaszanie emisji reszt tryptofanowych enzymu: Trp38, Trp55, Trp88 oraz Trp223 jonami P. Pomiary naturalnej fluorescencji lipazy Rml wykonywane były w buforowanym roztworze wodnym o pH 7.2 dla zakresu długości fal wzbudzenia: 290 - 300 nm. Wyniki badań wygaszania fluorescencji enzymu wskazują na obecność dwóch frakcji reszt tryptofanowych: wygaszalnej jonami I-, której udział w całkowitej fluorescencji Rml wzbudzanej przy λex = 295 nm wynosi 51% zaś stała Sterna-Volmera Ksv - 9.45 ± 0.9 M-1 oraz niewygaszalnej jonami I-, stanowiącej 49% całkowitej emisji białka lipazy. Analiza krzywych wygaszania zarejestrowanych w zakresie długości fal emisji: 320 - 380 nm pozwala na stwierdzenie, iż położenie maksimum fluorescencji wygaszalnej frakcji reszt tryptofanowych lipazy zależy od długości fali wzbudzenia i przypada na: 344, 348, 349 oraz 354 ± 1 nm odpowiednio dla λex = 290, 295, 297 oraz 300 nm. Podobnie, wraz ze zmianą długości fali wzbudzenia obserwuje się wzrost wartości stałej wygaszania dynamicznego od 8.00 ± 0.43 M-1 do 11.90 ± 0.49 M-1. Maksimum reszt tyrptofanowych niedostępnych dla zastosowanego wygaszacza nie zależy od długości fali wzbudzenia i przypada na 338 ± 1 nm. Obliczone rozkłady spektralne oraz stałe Sterna-Volmera wygaszalnej frakcji chromoforów lipazy Rml wskazują na hydrofilowy charakter mikrootoczenia reszt tryptofanowych oraz znaczny stopień ekspozycji reszt do rozpuszczalnika. Nie wykluczone, że reszty (bądź reszta) tryptofanowe dostępne dla wygaszacza zlokalizowane są tuż przy powierzchni molekuły białkowej i w znacznym stopniu związane są z emisją pochodzącą od Trp88. Chromofor ten występuje w krótkim, α-helikalnym odcinku łańcucha polipeptydowego lipazy Rml (reszty: 85-91). Fragment ten, zwany "wieczkiem", bierze bezpośredni udział w aktywacji enzymu.

2

Interaction of three Caenorhabditis elegans isoforms of translation initiation factor eIF4E with mono- and trimethylated mRNA 5' cap analogues.

88%

Stachelska A. , Wieczorek Z. , Ruszczyńska K. , Stolarski R. , Pietrzak M. , Lamphear B. J. , Rhoads R. E. , Darżynkiewicz E. , Jankowska-Anyszka M.

Acta Biochimica Polonica

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2002

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tom 49

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nr 3

671-682

EN

Translation initiation factor eIF4E binds the m7G cap of eukaryotic mRNAs and mediates recruitment of mRNA to the ribosome during cap-dependent translation initiation. This event is the rate-limiting step of translation and a major target for translational control. In the nematode Caenorhabditis elegans, about 70% of genes express mRNAs with an unusual cap structure containing m32,2,7G, which is poorly recognized by mammalian eIF4E. C. elegans expresses five isoforms of eIF4E (IFE-1, IFE-2, etc.). Three of these (IFE-3, IFE-4 and IFE-5) were investigated by means of spectroscopy and structural modelling based on mouse eIF4E bound to m7GDP. Intrinsic fluorescence quenching of Trp residues in the IFEs by iodide ions indicated structural differences between the apo and m7G cap bound proteins. Fluorescence quenching by selected cap analogues showed that only IFE-5 forms specific complexes with both m7G- and m32,2,7G-containing caps (Kas 2×106 M-1 to 7×106 M-1) whereas IFE-3 and IFE-4 discriminated strongly in favor of m7G-containing caps. These spectroscopic results quantitatively confirm earlier qualitative data derived from affinity chromatography. The dependence of Kas on pH indicated optimal cap binding of IFE-3, IFE-4 and IFE-5 at pH 7.2, lower by 0.4 pH units than that of eIF4E from human erythrocytes. These results provide insight into the molecular mechanism of recognition of structurally different caps by the highly homologous IFEs.

3

Fluorescence imaging of hybrid nanostructures composed of natural photosynthetic complexes and reduced graphene oxide

75%

Twardowska M. , Chomicki D. , Kamińska I. , Niedziółka-Jönsson J. , Maćkowski S.

Nanospectroscopy

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2014

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tom 1

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nr 1

EN

Herein, we describe the results of fluorescence microscopy imaging of peridinin-chlorophyll-protein (PCP) photosynthetic complex mixed with reduced graphene oxide (rGO). Upon incorporation of rGO the fluorescence image of PCP changes substantially from one characterized by uniform intensity towards a more complex pattern. The isolated bright spots feature up to ten times higher emission intensity compared to the fluorescence of PCP in the reference sample, where no rGO was added. The number of the bright spots increases with increasing rGO concentration. At the same time the fluorescence intensity away from the bright spots in the PCP/rGO hybrid system is quenched in comparison to the PCP – only reference.

4

In vitro fluorescence studies of transcription factor IIB-DNA interaction

75%

Górecki A. , Figiel M. , Dziedzicka-Wasylewska M.

Acta Biochimica Polonica

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2015

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tom 62

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nr 3

413-421

EN

General transcription factor TFIIB is one of the basal constituents of the preinitiation complex of eukaryotic RNA polymerase II, acting as a bridge between the preinitiation complex and the polymerase, and binding promoter DNA in an asymmetric manner, thereby defining the direction of the transcription. Methods of fluorescence spectroscopy together with circular dichroism spectroscopy were used to observe conformational changes in the structure of recombinant human TFIIB after binding to specific DNA sequence. To facilitate the exploration of the structural changes, several site-directed mutations have been introduced altering the fluorescence properties of the protein. Our observations showed that binding of specific DNA sequences changed the protein structure and dynamics, and TFIIB may exist in two conformational states, which can be described by a different microenvironment of W52. Fluorescence studies using both intrinsic and exogenous fluorophores showed that these changes significantly depended on the recognition sequence and concerned various regions of the protein, including those interacting with other transcription factors and RNA polymerase II. DNA binding can cause rearrangements in regions of proteins interacting with the polymerase in a manner dependent on the recognized sequences, and therefore, influence the gene expression.

5

Experimental and theoretical investigation of drotaverine binding to bovine serum albumin

63%

Gałęcki K. , Courtade G. , McFall A. , Prieschl Teixeira R. , Grgic M. , Esteve-Sistere M. , Maglemose Westphalen R. , Kowalska-Baron A.

Biotechnology and Food Science

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2013

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tom Vol. 77, nr 1

25--36

EN

This study was motivated by the need to provide more insight into the possible mechanism of the intermolecular interactions between antispasmodic drug drotaverine and one of the serum albumins (BSA), with the aim to indicate the most probable sites of these interactions. For this purpose both experimental (spectrofluorometric titration at various temperatures) and theoretical (molecular mechanics) methods have been applied. The obtained results clearly showed that drotaverine quenched BSA fluorescence, and the most probable mechanism is static quenching. The negative value of the theoretically predicted binding free Gibbs energy (-23.8 kJ/mol) confirmed the existence of the intermolecular interactions involving drotaverine and one tryptophan within BSA protein and was well agreed with the experimentally determined value of -25.2 kJ/mol.

6

Determination of trace amount of nitrite by fluorescent quenching method with phenosafranine enhanced by ß-cyclodextrin and its derivatives

63%

Jiang Z. T. , Guo Y. X. , Li R.

Chemia Analityczna

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2008

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tom Vol. 53, No. 4

569-582

EN

A new fluorescent quenching method has been developed for the determination of nitrite in food and water samples. The method is based on the reaction between nitrite and pheno-safranine (PS), which is used as an emission reagent and can react with nitrite to form a weakly-fluorescent compound in 0.02 mol L-1 hydrochloric acid. The sensitivity is largely enhanced in the presence of cyclodextrins (CDs) because of complexation. The effect of β-cyclodextrin (β-CD) and its three derivatives was compared and HP-p-cyclodextrin (HP-p-CD) was most effective. The excitation and emission of fluorescence were at wavelengths 530 and 608 nm, respectively. In the presence of HP-β-CD, the relationship between the fluorescence intensity and nitrite concentration was obtained up to 2.2 μg ml-1. The detection limit was 0.90 ng ml-1. hi the presence of 20 coexistent ions no serious interferences for most ions were observed. The method has been applied to determine nitrite in meat products, potato and water samples with satisfactory results.

PL

Opracowano nową metodę polegającą na tłumieniu fluorescencji azotanów(III) w próbkach wody i żywności. Metoda polega na reakcji azotanów(IH) z fenosafraniną (PS), która jest stosowana jako odczynnik powodujący fluorescencję reagujący z azotanami(III) osłabiającymi fluorescencję w 0,02 mol L-1 roztworze kwasu chlorowodorowego. Czułość reakcji znacznie się zwiększa w obecności cyklodekstryn (CD) w wyniku reakcji kompleksowania. Porównano efekt działania β-cyklodekstryny (β-CD), i trzech jej pochodnych. Najskuteczniejsze działanie zaobserwowano w obecności hydroksypropylo-p-cyklodekstryny (HP-β-CD). Długość fali promieniowania wzbudzającego i emitowanego wynosiła odpowiednio 539 i 608 nm. W obecności HP-β-CD uzyskano liniową zależność natężenia fluorescencji od stężenia azotanów(III) w zakresie do 2,2 μg. Granica wykrywalności wynosiła 0,9 ng mL-1. Nie obserwowano interferencji w obecności większości obcych jonów. Metodę zastosowano z powodzeniem do oznaczania azotanów (III) w próbkach produktów mięsnych, ziemniaków i wody.

7

Sensitive fluorimetric determination of protein with dibromohydroxyphenylfluorone-molybdenum(VI) comlex probe based on fluorescence quenching in non-ionic microemulsion

63%

Wei Q. , Li Y. , Yan T. , Duan C. , Ma H. , Bin D.

Chemia Analityczna

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2006

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tom Vol. 51, No. 5

691-700

EN

In this paper binding interaction between a new fluorescence probe dibromohydroxyphenyi-fluoronc-molybdenum(VI) (DBHPF-Mo(Vl) and a protein has been studied. In the presence of the cmulsifier OP microemulsion of pH 3.2 DBHPF-Mo(VI) complex binds the protein rapidly to form a stable compound of a maximum excitation wavelength (λex) of 468 nm and a maximum emission wavelength (λcm) of 527 nm. Fluorescence intensity of the probe is quenched by the protein. The magnitude of quenching is linearly proportional to the protein concentration. Under optimum conditions calibration plots for bovine serum albumin (BSA), human serum albumin (USA), and ovalburnin (Ova) have been constructed in the concentration ranges of 0∼6.00 μg mL-1, 0∼4.00 μg mL-1 and 0∼4.00 -1 , respectively. Addition of the OP microemulsion to the system during protein determination has increased significantly the sensitivity of the analysis by changing the microenvi-ronment. The corresponding detection limits of BSA, HSA and Ova were 3.4 ng mL-1, 3.6 ng mL-1 and 6.2 ng mL-1 The developed procedure has been satisfactorily applied to the determination of protein in urine.

PL

Zbadano oddziaływanie nowego odczynnika fluorescencyjnego: dibromohydroksyfluoron-molibden(VJ) (DBHPF-Mo(VI) z białkiem, W obecności emulgatora OP, przy pH 3,2 mikroemulsja kompleksu DBHPF-Mo(Vl) wiąże szybko białko, tworząc trwale połączenie o długości fali promieniowania wzbudzającego 468 nm i emitowanego 527 nm. Intensywność fiuorescencj i jest tłumiona w obecności białka. Wielkość tłumienia jest liniowo proporcjonalna do stężenia białka. W optymalnych warunkach otrzymano wykresy kalibracyjne dla albuminy surowicy wołowej (BSA), albuminy surowicy ludzkiej (HSA) i owalbuminy (Ova) w zakresach odpowiednio: 0∼6.00 μg mL-1, 0∼4.00 μg mL-1. Dodatek emulgatora OP zwiększa znacznie czułość metody. Granice wykrywalności BSA. HSA i Ova wy noszą odpowiednio mL-1, 3.6 ng mL-1 and 6.2 ng mL-1 . Opracowaną procedurę zastosowano do oznaczania białka w moczu.

8

The study of phenylboronic acid optical properties towards creation of a glucose sensor

63%

Kur-Kowalska K. , Przybyt M. , Miller E.

Biotechnology and Food Science

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2014

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tom Vol. 78, nr 2

101--110

EN

The article presents influence of pH and glucose concentration on phenylboronic acid (PBA) fluorescence studied by steady-state and time-resolved measurements. Fluorescence of PBA decreases with growing pH. These changes reflected acid-base equilibrium of PBA and allowed to estimate value of pKd as 9.2, which is comparable with literature data. Fluorescence intensity of phenylboronic acid is quenched in presence of glucose. The effect of quenching is more pronounced with increasing pH. At pH 7 quenching can be described by Stern-Volmer equation, at pH 8 and 9 by modified one. The obtained quenching constants are growing with pH increase. The quenching of phenylboronic acid fluorescence by glucose is a static one, which is confirmed by time-resolved measurements. Two lifetimes were found for fluorescence decay of phenylboronic acid. The lifetimes are practically independent on pH and glucose concentration and also fraction of both lifetimes are nearly the same. The obtained Stern-Volmer constants can be interpreted as apparent equilibrium constants of ester formation between acid and glucose.

9

g-C3N4/ Bio–synthesized silver nanoparticle for fluorometric bio-sensing of acetylcholinesterase and malathion

51%

Ali A. H.

Advances in Materials Science

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2022

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tom Vol. 22, nr 3(73)

23--40

EN

Malathion is widely used in agriculture due to their high efficiency as insecticides. They are very toxic hazardous chemicals to both human health and environment even at low concentration. The detection of pesticides (malathion) at the low levels developed by the environmental protection agency (EPA) still remains a challenge. A highly efficient fluorescent biosensor based on g-C3N4/AgNPs for AChE and malathion detection is successfully developed by impregnation method. The structural and morphological properties of the nanocomposites were characterized by using powder X-ray diffraction (XRD), fourier- transform infrared spectroscopy (FT-IR) and scanning electron microscopy (SEM). The analysis confirmed that there is a strong interfacial interaction between g-C3N4 and AgNPs. The fluorescent responses show an increase in intensity upon the additions of AChE which indicates that AChE as enzyme was hydrolyzing the substrate ACh, with the increase in oxidative electron as the preferred route of reaction. The developed OFF-ON sensor immobilizes by Actylcholestrase (AChE) and use as new probe for malathion detection. In the absence of malathion, AChE−g-C3N4/AgNCs exhibit high fluorescence intensity. However, the strong interaction of the basic sites to malathion, causes fluorescence quenching via static quenching and Ag form aggregation on the surface of g-C3N4. The experimental parameter such as pH of buffer (pH=6), concentration of acetylcholine (1 mM) and malathion (500 μM) were optimized. The sensor was also more sensitive with Stern-Volmer quenching constants (KSV) of 3.48x10 3 M -1. The practical use of this sensor for malathion determination in Khat was also demonstrated. The obtained amount of malathion in Khat is 168.8 μM.

10

Determination of manganese in food by polymer-phase fluorescence quenching method with eosin in the presence of phenanthroline

51%

Jiang Z. T. , Li R.

Chemia Analityczna

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2008

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tom Vol. 53, No. 5

659-671

EN

A sensitive and selective polymer-phase fluorescence quenching method for determination of trace amounts of manganese in food samples has been developed. The method is based on the reaction between manganese and eosin in the presence of phenanthroline to form a weak-fluorescence compound at pH 8.2. The fluorescence intensity was measured in 5 mm quartz cells at excitation and emission wavelengths of 533 and 550 nm, respectively. There was a satisfactory linear relationship between the fluorescence quenching and concentration of manganese in the range 0.2-3.5 μg in 25 mL of sample solution (i.e. 8-140 μg L-1). Detection limit was 0.03 μg for 25 mL of sample solution and RSD was 1.9%. Coexisting ions did not interfere with the reaction of eosin with manganese. The proposed method was applied to the determination of trace amounts of manganese in millet, black rice, and tea with satisfactory results.

PL

Opracowano czułą i selektywną metodę gaszenia fluorescencji w fazie polimerowej do oznaczania śladowych ilości manganu w żywności. W metodzie wykorzystano reakcję manganu z eozyną w obecności renantroliny. w wyniku której powstaje przy pH 8,2 słabo fluoryzujący związek. Intensywność fluorescencji mierzono w 5 mm komórce kwarcowej przy dł. fal wzbudzenia i emisji wynoszących odpowiednio 533 i 550 nm. Otrzymano dobrą zależność liniową między gaszeniem fluorescencji i stężeniem manganu w zakresie 0.2-3.5 μg w 25 mL roztworu próbki (czyli 8-140 μ g L-1).Wykrywalność wynosiła 0.03 μ g w 25 mL roztworu próbki, a względne odchylenie standardowe - l ,9%. Współ-występujące jony nie przeszkadzały w reakcji eozyny z manganem. Zaprapowaną metodę zastosowano do oznaczania śladowych ilości manganu w prosie, czarnym ryżu i w herbacie uzyskując dobre rezultaty.

11

Badania FTIR-ATR i fluorescencyjne układów białkowo-lipidowych

44%

Litwińczuk-Mammadova A. , Cieślik-Boczula K. , Rospenk M.

Wiadomości Chemiczne

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2017

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tom [Z] 71, 1-2

109--132

EN

Lipid-protein systems paly curtail roles in living systems [49]. Hence, a determination of their structure at different levels of organization is still one of the most important tasks in many research projects. A study of lipid-protein systems is based on many physicochemical techniques, such as spectroscopy of FTIR, Raman, fluorescence, NMR, EPR, as well as DLS, DSC and TEM methods. In the presented paper tow of the most frequently used methods, that is FTIR and fluorescence spectroscopy, will be discussed in details. They are characterized by a relatively low cost of sample preparation, a short measuring time, and they give a huge number of structural and physicochemical information about lipid-protein systems. In the FTIR-ATR spectroscopy many of vibrational bands are commonly used as very precise vibrational indicators of structural changes in lipids and proteins (Fig. 1) [1–6]. They allows to characterize lipid and protein components separately in mixed systems. Additionally, structural changes in lipid membranes can be monitored in one FTIR-ATR experiment simultaneously in a region of hydrophilic lipid head-groups (Fig. 5) [17, 18], in a hydrophobic part composed of hydrocarbon lipid chains (see Figures 2 and 3) [7–9], and in a lipid membrane interface represented by ester lipid groups (Fig. 4) [4, 6, 11, 12]. A secondary structure of proteins and peptides in different experimental conditions can be defined in the FTIR-ATR spectroscopy on the base of amide I bands (Fig. 6 and Tabs 1, 2 and 3) [20–22]. A fluorescence spectroscopy is a complementary methods to FTIR spectroscopy in a study of lipid-protein systems. It competes information about time-dependent and very fast (in a scale of femtoseconds) structural processes in both lipids [41–45] and proteins [23, 27, 48]. The folding, denaturation, and aggregation of proteins and lipid membranes accompanied by changes in an order, packing and hydration of the system under study [23, 27, 41–45, 48].