Interaction of lipoprotein lipase and apolipoprotein C-II with sonicated vesicles of 1,2-ditetradecylphosphatidylcholine: comparison of binding constants

The interaction of lipoprotein lipase (LpL) and its activator protein, apolipoprotein C-II (apoC-II), with a nonhydrolyzable phosphatidylcholine, 1,2-ditetradecyl-rac-glycero-3-phosphocholine (C14-ether-PC), was studied by fluorescence spectroscopy. A complex of 320 molecules of C14-ether-PC per LpL was isolated by density gradient ultracentrifugation in KBr. The intrinsic tryptophan fluorescence emission spectrum of LpL was shifted from 336 nm in the absence of lipid to 330 nm in the LpL-lipid complex; the shift was associated with a 40% increase in fluorescence intensity. Addition of C14-ether-PC vesicles to apoC-II caused a 2.5-fold increase in intrinsic tryptophan fluorescence and a shift in emission maximum from 340 to 317 nm. LpL and apoC-II/C14-ether-PC stoichiometries and binding constants were determined by measuring the increase in the intrinsic tryptophan fluorescence as a function of lipid and protein concentrations; for LpL the rate and magnitude of the fluorescence increases were relatively independent of temperature in the range 4-37 degrees C. A stoichiometry of 270 PC per LpL for the LpL-lipid complex compares favorably with the value obtained in the isolated complex. The dissociation constant (Kd) of the complex is 4.3 X 10(-8) M. For apoC-II, the stoichiometry of the complex is 18 PC per apoprotein, and the Kd is 3.0 X 10(-6) M. These data suggest that LpL binds more strongly than apoC-II to phosphatidylcholine interfaces.     

An unusual fluorescence spectrum of a protein proteinase inhibitor, Streptomyces subtilisin inhibitor.

Streptomyces subtilisin inhibitor, a dimeric protein proteinase inhibitor isolated in crystalline form by Murae et al. in 1972, contains three tyrosine and one tryptophan residues per monomer unit and has unusual fluorescence properties. When excited at 280 nm, it shows a characteristic fluorescence spectrum having a peak at 307 nm and a shoulder near 340 nm, a feature which has been recognized only for a very few cases in proteins containing both tryosine and tryptophan residues. When excited at 295 nm, at which tryrosine scarcely absorbs, the inhibitor shows an emission spectrum with a peak at 340 nm characteristic of a tryptophan residue. The emission with a peak at 307 nm is considered to arise from the tryrosine residues. The tryptophan quantum yield of Streptomyces subtilisin inhibitor excited at 295 nm is very small, indicating that the tryptophan florescence is strongly quenched in the native state of the inhibitor. Below pH 4 the peak of the fluorescence spectrum of the inhibitor excited at 280 nm shifts toward 340-350 nm with a concomitant increase in the quantum yield. The structural change induced by low pH seems to release the tryptophan fluorescence from the quenching.     

Physicochemical characterization of bovine retinal arrestin

The native conformation of bovine retinal arrestin has been characterized by a variety of spectroscopic methods. The purified protein gives rise to a near uv absorption band centered at 279 nm which results from the absorbance of its 14 tyrosine and one tryptophan residue. The extinction coefficient for this absorption band was determined to be 38.64 mM-1, cm-1 using the tyrosinate-tyrosine difference spectrum method; this extinction coefficient is ca. 17% lower than the previously reported value, and provides estimates of protein concentration which are in good agreement with estimates from the Bradford colorimetric assay. When native arrestin is purified to homogeneity, it displays a fluorescence spectrum which is dominated by tyrosine emission with no discernible contribution from tryptophan. Observation of the tyrosine-like fluorescence is dependent on the purity and structural integrity of the protein. Denaturation of arrestin by guanidine hydrochloride results in a diminution of tyrosine fluorescence and the concomitant appearance of a second fluorescence maximum at ca. 340 nm, presumably due to the single tryptophan residue. Thermal denaturation of arrestin leads to a conformation characterized by a broad fluorescence band centered at ca. 325 nm. Study of the arrestin fluorescence spectrum as a function of temperature indicates that the thermal denaturation is well modeled as a two-state transition with a transition midpoint of 60 degrees C. Temperature-dependent far uv circular dichroism studies indicate that changes in secondary structure occur coincident with the change in fluorescence. Studies of the temperature dependence of arrestin binding to light-adapted phosphorylated rhodopsin shows a strong correlation between the fluorescence spectral features of arrestin and its ability to bind rhodopsin. These data suggest that the relative intensities of tyrosine and tryptophan fluorescence are sensitive to the structural integrity of the native (i.e., rhodopsin binding) state of arrestin, and can thus serve as useful markers of conformational transitions of this protein. The lack of tryptophan fluorescence for native arrestin suggests an unusual environment for this residue. Possible mechanisms for this tryptophan fluorescence quenching are discussed.     

Motions studies of the human alpha 1-acid glycoprotein (orosomucoid) followed by red-edge excitation spectra and polarization of 2-p-toluidinylnaphthalene-6-sulfonate (TNS) and of tryptophan residues.

Dynamics studies on tryptophan residues of human alpha 1-acid glycoprotein (orosomucoid) and of 2-p-toluidinylnaphthalene-6-sulfonate bound to the protein are performed. Excitation at the red edge of the absorption spectrum of the tryptophan does not lead to a shift of the fluorescence emission maximum of the fluorophore. This reveals that Trp residues present motions with respect to their microenvironment. This is confirmed by polarization studies as a function of temperature. Excitation at the red edge of the absorption spectrum of TNS leads to an important shift (15 nm) of the fluorescence emission maximum of the probe. This reveals that emission of TNS occurs before relaxation of the amino-acids dipole occurs. Emission from a non-relaxed state means that TNS molecules are bound tightly to the protein, a result confirmed by polarization studies.     

Thermodynamic and kinetic studies of the interaction of vesicular dipalmitoylphosphatidylcholine with Agkistrodon piscivorus piscivorus phospholipase A2

The tryptophan fluorescence emission intensity at 340 nm of monomeric phospholipase A2 from Agkistrodon piscivorus piscivorus increased about 70% upon addition of dipalmitoylphosphatidylcholine small unilamellar vesicles (DPPC SUV) at 25 degrees C. The emission spectrum was also blue-shifted 6-8 nm, suggesting that the environment of 1 or more tryptophan residues had become less polar. This effect of SUV on the phospholipase A2 fluorescence was independent of Ca2+ at 25 degrees C, and the apparent association constant for the interaction was approximately 1.7 x 10(4) M-1. The apparent Km for hydrolysis of DPPC SUV was equal to the inverse of the estimated association constant. In the absence of Ca2+, the change in fluorescence intensity decreased with increasing temperature. Thermodynamic analysis of this reversible, temperature-dependent fluorescence change indicated that the A. p. piscivorus monomer phospholipase A2 interacts only with SUV in the true gel phase existing below the pretransition of gel to "ripple" phase lipid in the absence of Ca2+. In contrast, the fluorescence intensity change upon addition of SUV in the presence of Ca2+ was independent of temperature over the range of 25-48 degrees C. Under these conditions, hydrolysis of the lipid occurred concomitantly with the change in fluorescence which could not be reversed by the addition of EDTA. With a nonhydrolyzable analog of DPPC, however, the fluorescence changes upon mixing of SUV, Ca2+, and phospholipase A2 were reversible and temperature-dependent. Thus, the apparent irreversibility of the change in fluorescence observed with Ca2+ and DPPC SUV was correlated with hydrolysis of the vesicles. These results indicate that the magnitude of the initial interaction of enzyme with substrate is reversible, is Ca2+-independent, depends upon the lipid state, and is quantitatively correlated to the maximum rate of hydrolysis.     

Effect of temperature on tryptophan fluorescence of beta-lactoglobulin B.

The effect of heat on the conformation of bovine beta-lactoglobulin has been studied using intrinsic fluorescence spectroscopy. Changes in the intensity, wave-length of maximum emission and emission peak width at half height of tryptophan fluorescence over the range 15-90 degrees C at pH 6.4-6.5 has allowed the environments of the two tryptophans in the molecule to be discriminated. At 20 degrees C both tryptophans are in hydrophobic environments. As the temperature is raised the conformation changes such that at about 50 degrees C one of the tryptophans is transferred to a more polar environment accessible to solvent. Conformational changes appear to be reversible if the protein is cooled to 20 degrees C after heat treatments up to 70 degrees C. Above 70 degrees C the second tryptophan residue becomes exposed to solvent. Complete exposure of one residue occurs at 80 degrees C while the other is still partially buried even at 90 degrees C. When the protein is then cooled to 20 degrees C the conformational changes appear to be irreversible with only one tryptophan residue returning to the hydrophobic interior of the molecule.     

Tryptophan fluorescence studies of plasma fibronectin: effects of environmental factors

Steady state fluorescence measurements have been used to study tryptophan fluorescence of plasma fibronectin. The native protein has an emission maximum at 337 nm with a quantum yield of 0.03. A red shift of emission maximum was observed in 3–5M urea and a further red shift in 7–8M urea. The emission maximum shifted from 337 to 345 nm when the temperature was changed from 30 to 80°C, with a midpoint of thermal denaturation at 58°C. Similarly, the emission maximum shifted from 337 to 345 nm when the solution pH was increased from 9 to 12, with a midpoint of pH transition at 10.6. The results obtained from difference absorption spectroscopy studies suggest that the unfolding of fibronectin at alkaline pH is related at least in part to ionization of tyrosine residues. Since most of the tryptophan residues are in invariant positions in homology sequences, it is suggested here that tryptophan residues are useful intrinsic probes for elucidating fibronectin structure in solution.     

Porcine pancreatic alpha amylase and its isoforms--effect of deglycosylation by peptide-N-glycosidase F

Porcine pancreatic alpha-amylase (PPA) and its isoforms (PPA-I and PPA-II) were deglycosylated by peptide-N-glycosidase F (PNGase F) to investigate the role of bound carbohydrate. On deglycosylation, the effect on thermal stability was less pronounced. Deglycosylation resulted in a shift of the mid-point of thermal transition by 1-2 degrees C towards lower temperature. The fluorescence emission maxima of PPA, PPA-I and PPA-II were found to be 340nm indicating the presence of tryptophan residues in a fairly hydrophilic environment. A red shift in emission spectra accompanied by an increase in fluorescence intensity was observed upon deglycosylation.     

Fluorescence emission spectra of photosystem I, photosystem II and the light-harvesting chlorophyll a/b complex of higher plants

Fluorescence emission spectra excited at 514 and 633 nm were measured at -196 degrees C on dark-grown bean leaves which had been partially greened by a repetitive series of brief xenon flashes. Excitation at 514 nm resulted in a greater relative enrichment of the 730 nm emission band of Photosystem I than was obtained with 633 nm excitation. The difference spectrum between the 514 nm excited fluorescence and the 633 nm excited fluorescence was taken to be representative of a pure Photosystem I emission spectrum at -196 degrees C. It was estimated from an extrapolation of low temperature emission spectra taken from a series of flashed leaves of different chlorophyll content that the emission from Photosystem II at 730 nm was 12% of the peak emission at 694 nm. Using this estimate, the pure Photosystem I emission spectrum was subtracted from the measured emission spectrum of a flashed leaf to give an emission spectrum representative of pure Photosystem II fluorescence at -196 degrees C. Emission spectra were also measured on flashed leaves which had been illuminated for several hours in continuous light. Appreciable amounts of the light-harvesting chlorophyll a/b protein, which has a low temperature fluorescence emission maximum at 682 nm, accumulate during greening in continuous light. The emission spectra of Photosystem I and Photosystem II were subtracted from the measured emission spectrum of such a leaf to obtain the emission spectrum of the light-harvesting chlorophyll a/b protein at -196 degrees C.     

Tryptophan interactions with glycerol/water and trehalose/sucrose cryosolvents: infrared and fluorescence spectroscopy and ab initio calculations

In order to correlate how the solvent affects emission properties of tryptophan, the fluorescence and phosphorescence emission spectra of tryptophan and indole model compounds were compared for solid sugar glass (trehalose/sucrose) matrix and glycerol/water solution and under the same conditions, these matrices were examined by infrared spectroscopy. Temperature was varied from 290 to 12 K. In sugar glass, the fluorescence and phosphorescence emission spectra are constant over this temperature range and the fluorescence remains red shifted; these results are consistent with the static interaction of OH groups with tryptophan in the sugar glass. In sugar glass containing water, the water retains mobility over the entire temperature range as indicated by the HOH infrared bending frequency. The fluorescence of tryptophan in glycerol/water shifts to the blue as temperature decreases and the frequency change of the absorption of the HOH bend mode is larger than in the sugar glass. These results suggest rearrangement of glycerol and water molecules over the entire temperature change. Shifts in the fluorescence emission maximum of indole and tryptophan were relatively larger than shifts for the phosphorescence emission-as expected for the relatively smaller excited triplet state dipole for tryptophan. The fluorescence emission of tryptophan in glycerol/water at low temperature has maxima at 312, 313, and 316 nm at pH 1.4, 7.0, and 10.6, respectively. The spectral shifts are interpreted to be an indication of a charge, or Stark phenomena, effect on the excited state molecule, as supported by ab initio calculations. To check whether the amino acid remains charged over the temperature range, the infrared spectrum of alanine was monitored over the entire range of temperature. The ratio of infrared absorption characteristic of carboxylate/carbonyl was constant in glycerol/water and sugar glass, which indicates that the charge was retained. Tryptophan buried in proteins, namely calcium parvalbumin from cod and aldolase from rabbit, showed temperature profiles of the fluorescence spectra that were largely independent of the solvent (glycerol/water or sugar glass) and temperature whereas the fluorescence and phosphorescence yields were dependent. The results demonstrate how the rich information found in tryptophan luminescence can provide information on the dipolar nature and dynamics of the matrix.     

The ultraviolet fluorescence spectra of DPN-linked isocitrate dehydrogenase from bovine heart.

The emission maximum of DPN-linked isocitrate dehydrogenase from bovine heart shifted from 316 nm to 324 nm as the excitation wavelength was varied from 265 nm to 300 nm. This shift was accompanied by a nonproportional change in fluorescence intensity. Comparisons of the emission spectra of model compounds in aqueous buffer at pH 7.07 and n-butanol showed that lowered solvent polarity led to a blue shift of the peak of free tryptophan without significant change of fluorescence intensity, whereas the fluorescence intensity of tyrosine amide increased markedly without change in emission maximum. The emission peak of mixtures of tryptophan and tyrosine amide shifted to shorter wavelengths as the proportion of tyrosine amide increased. The results suggest a major contribution of tyrosine to the overall fluorescence of the dehydrogenase. DPNH caused quenching and a blue shift of the protein fluorescence maximum when excited between 270 nm and 290 nm, indicating that the two tryptophan residues per subunit of enzyme are located in different microenvironments of the protein and that DPNH may interact preferentially with the residue emitting at the longer wavelength.     

Development of an optical sensor for chlortetracycline detection based on the fluorescence quenching of l‐tryptophan

A simple and selective spectrofluorimetric method for the detection of chlortetracycline (CTC) was studied. In pH 7.4 buffer medium l ‐tryptophan (l ‐Trp), applied as the fluorescence probe, interacted with CTC resulting in fluorescence quenching of the probe. CTC was detected with maximum excitation and emission wavelengths at λ_ex/λ_em = 275/350 nm. Notably, quenching of fluorescence intensities was positively proportional to the CTC concentration over the range of 0.65–30 μmol L^?1 and the limit of detection was 0.2 μmol L^?1. Effect of temperature shown in Stern?Volmer plots, absorption spectra and fluorescence lifetime determination, indicated that fluorescence quenching of l ‐Trp by CTC was mainly by static quenching. The proposed study used practical samples analysis satisfactorily.     

Time-resolved fluorescence spectroscopy of human adenosine deaminase: effects of enzyme inhibitors on protein conformation

Adenosine deaminase, a purine salvage enzyme essential for immune competence, was studied by time-resolved fluorescence spectroscopy. The heterogeneous emission from this four-tryptophan protein was separated into three lifetime components: tau 1 = 1 ns and tau 2 = 2.2 ns an emission maximum at about 330 nm and tau 3 = 6.3 ns with emission maximum at about 340 nm. Solvent accessibility of the tryptophan emission was probed with polar and nonpolar fluorescence quenchers. Acrylamide, iodide, and trichloroethanol quenched emission from all three components. Acrylamide quenching caused a blue shift in the decay-associated spectrum of component 3. The ground-state analogue enzyme inhibitor purine riboside quenched emission associated with component 2 whereas the transition-state analogue inhibitor deoxycoformycin quenched emission from both components 2 and 3. The quenching due to inhibitor binding had no effect on the lifetimes or emission maxima of the decay-associated spectra. These observations can be explained by a simple model of four tryptophan environments. Quenching studies of the enzyme-inhibitor complexes indicate that adenosine deaminase undergoes different protein conformation changes upon binding of ground- and transition-state analogue inhibitors. The results are consistent with localized structural alterations in the enzyme.     

Spectral properties and function of two lumazine proteins from Photobacterium

The spectral properties are compared for two 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, one from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi. The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the two proteins: at 2 degrees C and 50 mM Pi, pH 7, fluorescence quantum yield phi F = 0.59 and 0.54, respectively; fluorescence lifetime tau = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 X 10(3) deg cm2 dmol-1. The ligand binding sites therefore must provide very similar environments, and arguments are presented that the bound ligand is relatively exposed to solvent. The dissociation equilibrium was studied by steady-state fluorescence polarization. The thermophilic protein binds the ligand with Kd (20 degrees C) = 0.016 microM, 10 times more tightly than the other protein [Kd (20 degrees C) = 0.16 microM]. The origin of the binding difference probably resides in differences in secondary structure. The tryptophan fluorescence spectra of the two proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy. For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very "floppy" region of the protein. For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Frequency-domain fluorescence studies of an extracellular metalloproteinase of Staphylococcus aureus

A frequency-domain fluorescence study of calcium-binding metalloproteinase from Staphylococcus aureus has shown that this two-tryptophan-containing protein exhibits a double-exponential fluorescence decay. At 10 degrees C in 0.05 M Tris-HCl buffer (pH 9.0) containing 10 mM CaCl2, fluorescence lifetimes of 1.2 and 5.1 ns are observed. Steady-state and frequency-domain solute-quenching studies are consistent with the assignment of the two lifetimes to the two tryptophan residues. The tryptophan residue characterized by a shorter lifetime has a maximum of fluorescence emission at about 317 nm and the second one exhibits a maximum of its emission at 350 nm. These two residues contribute almost equally to the protein's fluorescence. These results, as well as fluorescence-quenching studies using KI and acrylamide as a quencher, indicate that in calcium-loaded metalloproteinase, the tryptophan residue characterized by the shorter lifetime is extensively buried within the protein. The second residue is exposed on the surface of the protein. The tryptophan residues of metalloproteinase have acrylamide dynamic-quenching rate constants, kq values, of 2.3 and 0.26 X 10(9) M-1 X s-1 for the exposed and buried residue, respectively. A study of the temperature dependence of the fluorescence lifetime for the two tryptophan components gives activation energies, Ea values, for thermal quenching of 1.8 and 2.2 kcal/mol for the buried and the exposed residue, respectively. Dissociation of Ca2+ from the protein causes a change in the protein's structure, as can be judged from dramatic changes which occur in the fluorescence properties of the buried tryptophan residue. These changes include an approx. 13 nm red-shift in the maximum of the fluorescence emission and an increase in the acrylamide-quenching rate constant, and they indicate that the removal of Ca2+ results in an increase in the exposure and the polarity of the microenvironment of this 'blue' residue.     

Changes in protein conformation and stability accompany complex formation between human C1 inhibitor and C1-s

The fluorescence spectrum of C1 inhibitor (C1-Inh) in aqueous buffer has a maximum at 324 nm which shifts to 358 nm in 6.0 M guanidinium chloride (GdmC1), indicating that fluorescent tryptophans are buried in the native protein. When titrated with GdmC1, the fluorescence intensity, polarization, and emission maximum of C1-Inh and C1-s exhibited clear transitions which were more prominent than those of the enzyme-inhibitor complex. Two of the variables (intensity and emission maximum) suggest biphasic unfolding of C1-Inh. Differential absorption measurements and sodium iodide quenching of intrinsic fluorescence were consistent with a net increase in the exposure of tryptophans and tyrosines upon complex formation. This reaction, i.e., complex formation, was also accompanied by an increase in the ability to enhance the fluorescence of the hydrophobic probe 8-anilino-1-naphthalenesulfonate. Fluorescence assays of heat denaturation showed transitions at 40 and 52 degrees C for C1-s and at 60 degrees C for C1-Inh whereas there was no detectable melting transition for the complex. Similarly, differential scanning calorimetric measurements revealed transitions at 42, 52, and 62 degrees C for C1-s and one transition at 60 degrees C for C1-Inh, with no major transitions detectable for the complex. The ratio of the calorimetric enthalpy to the apparent van't Hoff enthalpy for thermal unfolding of C1-Inh was 1.6. Taken together, these results suggest that C1-Inh and C1-s are each composed of at least two independently unfolding domains and that complex formation, which involves conformational change, yields a protein substantially more stable than either component alone.     

Heme protein fluorescence versus pressure.

Fluorescence spectra of several ferric heme proteins have been measured vs. pressure to 6,000 bars. Sperm whale myoglobin (SW Mb), Aplysia myoglobin, leghemoglobin (Lb), and cytochrome P450 all show excitation and emission spectra characteristic of tryptophan in proteins with peak emission at 330-340 nm. At one bar, the fluorescence is weak due to energy transfer to the heme group, which makes the yield a sensitive probe of protein unfolding at high pressure. After an initial decrease of a few percent per kbar, the protein shows a large increase in fluorescence at high pressure. The increase is pH dependent and the results indicate that several high pressure states occur. For SW Mb at 15 degrees C an increase of a factor of 20 occurs with midpoint at 2,000 bars at pH 5 and is only partially reversible, while the increase at pH 7 occurs at 4,000 bars and is only half as large and is completely reversible. Aplysia Mb and Lb show a similar effect, but unfold at a higher pressure than SW Mb. P450 also shows a transition to a state of higher fluorescence, but the transition in this case is irreversible as a stable form, P420, is formed. The fluorescence intensity measurements permit an estimation of the increase in the TRY-heme distance in the high pressure state.     

Picosecond and steady state, variable intensity and variable temperature emission spectroscopy of bacteriorhodopsin.

The bacteriorhodopsin emission lifetime at 77 degrees K has been obtained for different regions of the emission spectrum with single-pulse excitation. The data under all conditions yield a lifetime of 60 +/- 15 ps. Intensity effects on this lifetime have been ruled out by studying the relative emission amplitude as a function of the excitation pulse energy. We relate our lifetime to previously reported values at other temperatures by studying the relative emission quantum efficiency as a function of temperature. These variable temperature studies have indicated that an excited state with an emission maximum at 670 nm begins to contribute to the spectrum as the temperature is lowered. Within our experimental error the picosecond data seem to suggest that this new emission may arise from a minimum of the same electronic state responsible for the 77 degrees K emission at 720 nm. A correlation is noted between a 1.0-ps formation time observed in absorption by Ippen et al. (Ippen, E.P., C.V. Shank, A. Lewis, and M.A. Marcus. 1978. Subpicosecond spectroscopy of bacteriorhodopsin. Science [wash. D.C.]. 200:1279-1281 and a time extrapolated from relative quantum efficiency measurements and the 77 degrees K fluorescence lifetime that we report.     

Luminescence studies of saccharide binding to wheat germ agglutinin (lectin).

The fluorescence and phosphorescence emission of wheat germ agglutinin are reported. Fluorescent tryptophan residues of wheat germ agglutinin are found highly exposed to solvent: fluorescence quenching induced by temperature fits with a single Arrhenius critical energy close to that of tryptophan in solution; the whole fluorescence emission is susceptible to iodide ion quenching and data reveal the homogeneity of fluorescence arising from only one type of tryptophan exposition. Energy transfers are analyzed at singlet and triplet state level. Tyrosine fluorescence at 25 degrees C is very weak. Results obtained from the relative excitation fluorescence quantum yield and from intrinsic fluorescence polarization show that a large amount of energy absorbed by tyrosine at 280 nm is transferred to tryptophan residues. However, tyrosine fluorescence is highly increased at 70 degrees C although disulfide bridges are not reduced. The phosphorescence spectrum at 77 K in 50% ethylene glycol is finely structured with several resolved vibrational bands at 405, 432 and 455 nm. Phosphorescence decay can be fitted with a single exponential. Lifetime is independent of excitation wave-length. Its value is very close to that of free tryptophan. Influence of tri-N-acetyl-chitotriose binding on luminescence properties are investigated. Results are analyzed in terms of steric tryptophan-ligand relationships. It is shown that all the fluorescent chromophores are concerned by the ligand binding but all fluorescence emission is still susceptible to iodide ion quenching. There is no change induced in energy transfer at the singlet state level and no modification in triplet state population.     

Evidence for the existence of an unfolding intermediate state for aminoacylase during denaturation in guanidine solutions

The equilibrium unfolding of pig kidney aminoacylase in guanidinium chloride (GdmCl) solutions was studied by following the fluorescence and circular dichroism (CD). At low concentrations of GdmCl, less than 1.0 M, the fluorescence intensity decreased with a slight red shift of the emission maximum (from 335 to 340 nm). An unfolding intermediate was observed in low concentrations of denaturant (between 1.2 and 1.6 M GdmCl). This intermediate was characterized by a decreased fluorescence emission intensity, a red-shifted emission maximum, and increased binding of the fluorescence probe 1-anilino-8-naphthalenesulfonate. No significant changes of the secondary structure were indicated by CD measurement. This conformation state is similar to a molten globule state which may exist in the pathway of protein folding. Further changes in the fluorescence properties occurred at higher concentrations of GdmCl, more than 1.6 M, with a decrease in emission intensity and a significant red shift of the emission maximum from 340 to 354 nm. In this stage, the secondary structure was completely broken. A study of apo-enzyme (Zn2+-free enzyme) produced similar results. However, comparison of the changes of the fluorescence emission spectra of native (Holo-) enzyme with Zn2+-free (Apo-) enzyme at low GdmCl concentrations showed that the structure of the Holo-enzyme was more stable than that of the Apo-enzyme.