Quenching of fluorescence by oxygen. A probe for structural fluctuations in macromolecules.
TL;DR: In this article, the fluorescence of various fluorophores by molecular oxygen has been studied in aqueous and nonaqueous solutions equilibrated with oxygen pressures up to 100 atm.
Abstract: Quenching of the fluorescence of various fluorophores by molecular oxygen has been studied in aqueous and nonaqueous solutions equilibrated with oxygen pressures up to 100 atm. Temperature dependence of quenching, agreement with the Stern–Volmer equation, and fluorescence lifetime measurements indicate that essentially all the observed quenching is dynamic and close to the diffusion-controlled limits. Studies of charged polyamino acids containing tryptophan show that oxygen quenching, in contrast to I−, is completely insensitive to charge effects. Ethidium bromide, when intercalated into double helical DNA, is quenched with 1/30th of the efficiency of the free dye in solution. Three dyes bound to bovine serum albumin were also found to be relatively protected from the free diffusion of oxygen. Quenching of intrinsic or bound fluorophores by molecular oxygen is therefore an appropriate method to determine the accessibility to oxygen of regions of the macromolecule surrounding the fluorophore and indirectly the structural fluctuations in the macromolecule that permit its diffusion to the fluorophore.
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TL;DR: A review of the use of the technique of solute fluorescence quenching to study the structure and dynamics of proteins and a number of factors are discussed that must be considered in analyzing such data.
1,644 citations
TL;DR: Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid.
Abstract: Though the structures presented in crystallographic models of macromolecules appear to possess rock-like solidity, real proteins and nucleic acids are not particularly rigid. Most structural work to date has centred upon the native state of macromolecules, the most probable macromolecular form. But the native state of a molecule is merely its most abundant form, certainly not its only form. Thermodynamics requires that all other possible structural forms, however improbable, must also exist, albeit with representation corresponding to the factor exp( — Gi/RT) for each state of free energy Gi (see Moelwyn-Hughes, 1961), and one appreciates that each molecule within a population of molecules will in time explore the vast ensemble of possible structural states.
1,261 citations
TL;DR: The current state of optical methods for sensing oxygen have become powerful alternatives to electrochemical detection and in the process of replacing the Clark electrode in many fields and a selection of specific applications of such sensors are given.
Abstract: We review the current state of optical methods for sensing oxygen. These have become powerful alternatives to electrochemical detection and in the process of replacing the Clark electrode in many fields. The article (with 694 references) is divided into main sections on direct spectroscopic sensing of oxygen, on absorptiometric and luminescent probes, on polymeric matrices and supports, on additives and related materials, on spectroscopic schemes for read-out and imaging, and on sensing formats (such as waveguide sensing, sensor arrays, multiple sensors and nanosensors). We finally discuss future trends and applications and summarize the properties of the most often used indicator probes and polymers. The ESI† (with 385 references) gives a selection of specific applications of such sensors in medicine, biology, marine and geosciences, intracellular sensing, aerodynamics, industry and biotechnology, among others.
847 citations
TL;DR: Quenching of the tryptophan fluorescence of proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pres- sure of 1500 psi, and revealed no significant perturbation of the protein structure.
Abstract: Quenching of the tryptophan fluorescence of na- tive proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pres- sure of 1500 psi. Measurement of absorption spectra and en- zymic activities of protein solutions under these conditions reveal no significant perturbation of the protein structure. The oxygen quenching constant (k+*) for a variety of proteins indi- cates that the apparent oxygen diffusion rate through the pro- tein matrix is 20-50z of its diffusion rate in water. No tryp- tophan residues appear to be excluded from quenching, and no correlation of the fluorescence emission maxima with k+* T he previous paper (Lakowicz and Weber, 1973) de- scribed the methodology and presented experimental data for the quenching of small molecules, and some linear biopoly- mers, by oxygen. Here we examine the quenching of the fluo- rescence of proteins by oxygen. X-Ray determined structures and solvent perturbation studies of many proteins have shown that tryptophan residues are often situated in the interior of the protein matrix and appear inaccessible to solvent. It must be pointed out, however, that both of these techniques yield information about the average conformation and solvation of the amino acid residues, but no information about the ex- istence of the structural fluctuations which may occur. Since quenching of fluorescence by oxygen depends on the colli- sional rate between oxygen and fluorophore, we expected oxygen quenching of tryptophan fluorescence in proteins to yield information on the dynamics of those structural changes in the nanosecond time scale that would allow diffusion of oxygen through the protein matrix. Such local fluctuations must be indispensable for the effective quenching of a fluoro- phore shown by X-ray structural studies to be out of direct contact with water. The quenching of the fluorescence of fluorophore residues forming part of a protein, or in general of a compact macro- molecule, presents many complexities that are absent in the case of an isolated fluorophore in solution. These complexities can best be examined by reference to the modified Stern- Volmer equation discussed in the previous paper
759 citations
TL;DR: The results of synchronous fluorescence spectra and UV-vis absorption spectra show that the conformation of bovine serum albumin has been changed, and the quenching mechanism of fluorescence of BSA by monoammonium glycyrrhizinate was discussed.
586 citations
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TL;DR: Evidence is presented that formation of this complex (complex I) is specific for base-paired regions either in DNA, RNA or RNA: DNA hybrids, and that the basis of this specificity is intercalation of the dye between base pairs.
2,229 citations
TL;DR: The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity.
Abstract: The effect of iodide on the tryptophyl fluorescence of model compounds and of lysozyme was studied in order to evaluate the factors that determine the use of iodide as a selective quencher of the fluorescence of tryptophyl side chains of proteins exposed to solvent. The results with the model compounds indicate the involvement of a collisional quenching mechanism due to the agreement with the Stern-Volmer law and the proportionality of the quenching constant with To7 for indole-3-acetamide. Bimolecular rate constants, k a , calculated from measured quenching constants using available lifetime data are equal to, greater than, or less than 4-6 X lo9 M-' sec-l for uncharged, positively charged, and negaI n a preliminary study it was shown that a large fraction of the tryptophyi fluorescence of lysozyme in aqueous solution was quenched by low concentrations of iodide ion (Lehrer, lY67). It was concluded from a study of the magnitude of the quenching of fluorescence and the character of the difference fluorescence spectrum produced in the presence and absence of substrate that the fluorescence of tryptophyls exposed to solvent and located in the substrate binding site was preferentially quenched by iodide. It appeared that this technique, which can be called solute perturbation of protein fluorescence, could be used as a probe of fluorophor exposure in proteins in a manner analogous to the technique of solvent perturbation of protein absorption (Herskovits and Laskowski, 1960; Laskowski, 1966). * From the Department of Muscle Research, Boston Biomedical Research Institute, Boston, Massachusetts 021 14, and from the Department of Neurology, Harvard Medical School, Boston, Massuchusetts 02115. Receired April 22, 1971. This work was supported by grants from the National Institutes of Health (AM 11677 and HE 0581 1) and the iMass'ichuserts Heart Association (516). tively charged tryptophyl compounds, respectively. A modified version of the Stern-Volmer law was calculated for a fluorophor population with different quantum yields and quenching constants. This formulation allows the calculation of the effective quenching constant from the intercept and the slope at low iodide concentration of a F o ] M cs. l/(I-) plot. Data obtained for lysozyme indicate that for the native protein about one-half the tryptophyl fluorescence is accessible at pH 5.3 whereas all of the tryptophyl fluorescence is accessible in 6 M G d n . HCI. Information regarding the presence of charged groups near tryptophyl side chains was obtained for lysozyme by studying the dependence of the quenching on pH. More recently, studies by other workers have ~ised bromate (Winkler, 1969) and iodide (Arrio er al., 1970) to quench extrinsic fluorescence (Teale and Badley, 1970). Oxygen has also been used as a quencher of pyrenebutyric acid bound to proteins (Vaughan and Weber, 1970). Burstein (1968a) has also independently studied the quenching of tryptophyl fluorescence in model compounds by iodide. In order to learn more about the quenching mechanism and the factors which determine fluorophor exposure, various tryptophyl model compounds and a model protein, lysozyme. were used in the present study. The results of the model compound study provide evidence for a mechanism that follows the classical Stern-Volmer law (1919), predominantly involving collisional quenching, and illustrate the importance of local charge and solvent viscosity. The quenching of lysozyme fluorescence by iodide also appears to follow a similar mechanism because of the agreement obtained with a inodified version of the Stern-Volmer law which was calculated for a heterogeneous distribution of fluorophors in a protein. Effective Stern-Volmer quenching constants and values for the fractional accessible fluorescence were obtained for lyso3254 B I O C H E M I S T R Y , V O L . 1 0 , N O . 1 7 , 1 9 7 1 I O D I D E Q U E N C H I N G O F P R O T E I N F L U O R E S C E N C E zyme in 6 M Gdn.HCI, 'S M urea, and in aqueous solution at different pH's using the modified Stern--Volmer law. Values obtained are consistent with information regarding accessibility obtained by other methods. Experimental Section Muteriais. The following high-purity compounds were used as obtained from Mann Research Laboratories, New York, N. Y. : indole-3-acetic acid, indole-3-propionic acid, indole-3-butyric acid, indole-3-acetamide, N-Ac-L-TrpNH?, L-TrpOEt, Gdn . HCI, and urea. L-Trp (Cyclo Chemical Corp., Los Angeles), KI , Na&03, citric acid, and NaCl (Fisher Scientific Co., Freehold, N. J.) were all of high purity and used as obtained. Indole (Fisher) and skatole (3-methylindole) (Mann) were recrystallized from methanol containing Norit A (Matheson Coleman & Bell, Rutherford, N. J.). Hepes buffer was used as obtained from Calbiochem (Los Angeles). Poly(Glug9Trp1) and poly(Lysg7Trp3) were high molecular weight random sequence copolymers kindly supplied by Dr. G. Fasman. Lysozyme from two different sources were used (twice crystallized from Worthington Biochemical Corp., Freehold, N. J., and six-times crystallized from Miles Laboratories, Elkhart, Ind.). Both preparations gave similar results. Ac3Glcn was kindly supplied by Dr. J. Rupley and glycol chitin was obtained from Miles Laboratories. Methods. Quenching measurements at constant pH were made on five solutions of a given material containing increasing amounts of K I (0-0.2 M). These were prepared by diluting stock solutions of the model compound, of KI, of NaC1, and of buffer, into volumetric flasks. NaCl was used to keep the ionic strength constant. Stock solutions of the indole compounds were used within a few days of preparation and kept in the dark at 0-5" overnight. A small amount of SO3?(ca. M) was added to the iodide solution to prevent 1 3 formation. This was necessary because Isabsorbs in the wavelength region of tryptophyl fluorescence (filter effect) and because of possible chemical reaction. The solutions were equilibrated at 25 O before the measurements. Stock solutions of lysozyme were routinely filtered through a Millipore filter (HAWP 0.45 p ) before use. pH titrations were performed in the absence and presence of iodide by adding small quantities of 0.5 M HC1 to the solution in the cuvet, which contained 2 mM Hepes and 2 mM citrate, originally pH 8, then measuring the pH and fluorescence. pH was measured with a Radiometer PHM4c meter standardized at pH 4 and 7. Fluorescence spectra and intensities were measured by exciting a t 280 nm or longer. In most cases no corrections for iodide absorption were necessary. The fluorescence of a reference (usually the 0.2 M NaC1-0.0 M K I solution) was measured just before measuring the fluorescence of each solution in order t o correct for any exciting lamp fluctuation. Fluorescence measurements were made with either an Aminco-Bowman spectrofluorometer or an instrument that employs two Jarrell-Ash 0.25-m monochromators, an EM1 9601 B photomultiplier, and either a high-pressure 200-W mercury lamp or a 150-W high-pressure xenon lamp. Low temperatures were obtained with a refrigerated water circulator attached to the sample housing. The temperature was measured by inserting a calibrated thermistor into the sample solution. Abbreviations used are: Gdn . HCI, guanidine hydrochloride; Trp, tryptophyl or tryptophan; Hepes, N-2-hydroxyethylpiperazine-N'2-ethanesulfonic acid ; Ac3GIcii. tri-N-acetyl-D-glucosamirie. The activity of lysozyme was determined by the method of Hamaguchi et a/ . (1960). The decrease in viscosity with time caused by hydrolysis of glycol chitin (2 mg/ml) by lysozyme (0.02 mg/ml) in the presence of 0.2 M NaCl or 0.2 M KI in 2 m M citrate (pH 5.5) is the basis of this method. The specific viscosity of glycol chitin solutions in Cannon viscometers at 25" was measured with time after a small volume of lysozyme was added. The slope of the approximately linear viscosity decrease between 1 and 10 min was used as a measure of activity. The optical rotatory dispersion and circular dichroism spectra of lysozyme (0.95 mg/ml) in 0.2 M NaCl or in 0.2 M KI , 2 mM citrate (pH 5.2) were measured in a 1-cm cell with a Jasco spectropolarimeter. The absorbance of Iprevented measurements below 265 nm. Difference spectra were either measured with a Cary 15 or a Beckman DK spectrophotometer using mixing cells (Pyrocell, Inc., N. Y . ) . The total absorption over the wavelengths scanned was always below 2.2. The low-temperature studies were performed with a Beckman D K using a refrigerated sample holder.
1,655 citations
1,173 citations
TL;DR: Quenching of the tryptophan fluorescence of proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pres- sure of 1500 psi, and revealed no significant perturbation of the protein structure.
Abstract: Quenching of the tryptophan fluorescence of na- tive proteins was studied using oxygen concentrations up to 0.13 M, corresponding to equilibration with oxygen at a pres- sure of 1500 psi. Measurement of absorption spectra and en- zymic activities of protein solutions under these conditions reveal no significant perturbation of the protein structure. The oxygen quenching constant (k+*) for a variety of proteins indi- cates that the apparent oxygen diffusion rate through the pro- tein matrix is 20-50z of its diffusion rate in water. No tryp- tophan residues appear to be excluded from quenching, and no correlation of the fluorescence emission maxima with k+* T he previous paper (Lakowicz and Weber, 1973) de- scribed the methodology and presented experimental data for the quenching of small molecules, and some linear biopoly- mers, by oxygen. Here we examine the quenching of the fluo- rescence of proteins by oxygen. X-Ray determined structures and solvent perturbation studies of many proteins have shown that tryptophan residues are often situated in the interior of the protein matrix and appear inaccessible to solvent. It must be pointed out, however, that both of these techniques yield information about the average conformation and solvation of the amino acid residues, but no information about the ex- istence of the structural fluctuations which may occur. Since quenching of fluorescence by oxygen depends on the colli- sional rate between oxygen and fluorophore, we expected oxygen quenching of tryptophan fluorescence in proteins to yield information on the dynamics of those structural changes in the nanosecond time scale that would allow diffusion of oxygen through the protein matrix. Such local fluctuations must be indispensable for the effective quenching of a fluoro- phore shown by X-ray structural studies to be out of direct contact with water. The quenching of the fluorescence of fluorophore residues forming part of a protein, or in general of a compact macro- molecule, presents many complexities that are absent in the case of an isolated fluorophore in solution. These complexities can best be examined by reference to the modified Stern- Volmer equation discussed in the previous paper
759 citations