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.     

New fluorescence of nonenzymatically glucosylated human serum albumin

Glucosylated human serum albumin (G-HSA) obtained under incubation with glucose at 37 degrees C for 8 days showed a new fluorescence with a maximum at 430 nm, resulting in quenching of the fluorescence of only one tryptophan residue on HSA. The quantum yield of new fluorescence is 0.024 at 25 degrees C. The analysis of the excitation spectra allowed us to conclude the absence of energy transfer. In G-HSA, non-disulfide cross-linking hexamer was confirmed by SDS-PAGE.     

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp.As temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp increases.The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to bRp.These results suggest that significant structural difference existed between bRband bRp.It was also found that both kinds of bR are highly thermal stable.The maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm formed.The transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high temperature.This phenomenon has not been reported before.     

Determination of dipicolinic acid in bacterial spores by derivative spectroscopy

Dipicolinic acid was extracted from approximately 0.1 mg spores or 0.5 ml of sporulating culture with 20 mM HCl for 10 min at 100 degrees C. The suspension was diluted with 5 mM Ca2+, 100 mM Tris, pH 7.6, centrifuged, and the first derivative of the uv absorbance spectrum recorded from 275 nm to 285 nm. DPA concentration was determined from the difference between the maximum at 276.6 nm and the minimum at 280 nm. The use of the difference between two first derivative values removed possible interference from sloping baselines. Turbidity, nucleic acids, and bacteriological media did not interfere. Analysis time for four extracts was 4 min using a spectrophotometer reading at 0.1-nm intervals. Dipicolinate at 0.1 mM gave 0.184 absorbance/nm at 25 degrees C. The coefficient of variation was 1.5%, and the detection limit 1 microM.     

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.     

Sulphation by cultured cells. Cysteine, cysteinesulphinic acid and sulphite as sources for proteoglycan sulphate.

The tryptophan-auxotrophic Bacillus subtilis LC33 mutant strain utilizes either tryptophan or 4-fluorotryptophan for growth. Proteins therefore could be isolated from these cells in either tryptophan-containing or 4-fluorotryptophan-containing forms. Since 4-fluorotryptophan is non-fluorescent, tryptophan fluorescence would be suppressed in the 4-fluorotryptophan-containing proteins, facilitating the investigation of other chromophores either on the proteins or interacting with the proteins. This approach, potentially applicable to any protein endogenous to or clonable into B. subtilis, was illustrated by an examination of the fluorescence of B. subtilis ribosomal proteins.     

Ultraviolet-Induced Photodegradation of Cucumber (Cucumis sativus L.) Microsomal and Soluble Protein Tryptophanyl Residues in Vitro

The in vitro effects of ultraviolet B (280-320 nm) radiation on microsomal membrane proteins and partially purified ribulose bisphosphate carboxylase (Rubisco) from cucumber (Cucumis sativus L.) was investigated by measuring the direct photolytic reduction of tryptophan fluorescence and the formation of fluorescent photooxidation products. Exposure of microsomes and Rubisco to monochromatic 300-nm radiation resulted in the loss of intrinsic tryptophan fluorescence and the production of blue-emitting fluorophores. The major product of tryptophan photolysis was tentatively identified as N-formylkynurenine (N-FK). Even though the rates of tryptophan photodegradation and N-FK formation were similar, the amount of blue fluorescence produced was significantly higher in the microsomes relative to Rubisco. Studies with various free radical scavengers and other modifiers indicated that tryptophan photodegradation requires oxygen and that the subsequent formation of N-FK may involve reactive oxygen species. The optimum wavelengths for loss of typtophan fluorescence were 290 nm for the microsomes and 280 nm for Rubisco. The temperature dependence of tryptophan fluorescence and rate of tryptophan photodegradation indicated an alteration in the cucumber microsomal membranes at about 24[deg]C, which influenced protein structure and tryptophan photosensitivity.     

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)     

Deuterium oxide stabilizes conformation of tubulin:a biophysical and biochemical study

The present study was aimed to elucidate the mechanism of stabilization of tubulin by deuterium oxide (D(2)O). Rate of decrease of tryptophan fluorescence during aging of tubulin at 4 degrees C and 37 degrees C was significantly lower in D(2)O than in H(2)O. Circular dichroism spectra of tubulin after incubation at 4 degrees C, suggested that complete stabilization of the secondary structure in D(2)O during the first 24 hours of incubation. The number of available cysteine measured by DTNB reaction was decreased to a lesser extent in D(2)O than in H(2)O. During the increase in temperature of tubulin, the rate of decrease of fluorescence at 335 nm and change of CD value at 222 nm was lesser in D(2)O. Differential Scanning calorimetric experiments showed that the T(m) values for tubulin unfolding in D(2)O were 58.6 degrees C and 62.17 degrees C, and in H(2)O those values were 55.4 degrees C and 59.35 degrees C.     

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.     

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.     

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.     

Picosecond energy transfer in Porphyridium cruentum and Anacystis nidulans.

Picosecond energy transfer is measured in Anacystis nidulans and Porphyridium cruentum. Fluorescence is sensitized by a 6-ps laser flash, at 530 nm. The time dependence of fluorescence is measured with reference to the laser pulse. Fluorescence is recorded from phycoerythrin (576 nm), R-phycocyanin (640 nm), allophycocyanin (666 nm), Photosystem II chlorophyll (690 nm) and long wave length chlorophyll (715 nm). Energy transfer measurements are made at 37 degrees C, 23 degrees C, and 0 degrees C, and 77 degrees K. It is shown that the rate of energy transfer can be varied with temperature. In both A. nidulans and P. cruentum there is a sequential transfer of excitation energy from phycoerythrin to phycocyanin to allophycocyan to Photosystem II chlorophyll fluorescence. The long wavelength chlorophyll fluorescence at 715 nm, however, does not always follow a sequential transfer of excitation energy. Depending on the temperature, fluorescence at 715 nm can precede fluorescence from phycocyanin.     

A Meccano set approach of joining trpzip a water soluble beta-hairpin peptide with a didehydrophenylalanine containing hydrophobic helical peptide.

A 16 residues long, water soluble, monomeric beta-hairpin peptide 'trpzip', stabilized by tryptophan zipper has been linked via a tetraglycyl linker to a hydrophobic didehydrophenylalnine (DeltaF) containing helical octapeptide. Circular dichroism studies of this 28 residues long peptide, 'trpzipalpha' (Ac-GEWTWDDATKTWTWTE-GGGG-DeltaFALDeltaFALDeltaFA-NH(2)) in water have revealed the presence of both the beta-hairpin and the helical conformations. This is the first instance where a DeltaF containing peptide has been found to display a helical fold in water. The fluorescence emission wavelengths of tryptophan in Ac-G-W-G-NH(2), trpzip and trpzipalpha were 341.5, 332.8 and 332.6 nm, respectively. The fluorescence quantum yield of trpzip was 2.6-fold higher than trpzipalpha suggesting that proximal interactions between the beta-hairpin and the helix caused the quenching of tryptophan fluorescence in the former by the DeltaFs in the latter. The molar ellipticity of the far UV couplet characteristic of trpzip was reduced in trpzipalpha and the CD based thermal melting temperatures at 228 nm were 62 degrees C (trpzip) and 57 degrees C (trpzipalpha). A concentration-dependent variable temperature CD study in water showed that in trpzipalpha, increasing temperature is detrimental to the beta-hairpin, but it augments the helical motif, perhaps by intermolecular oligomerization. Our results show that in water, trpzipalpha exhibits long-range interactions between two different secondary structures. In contrast to trpzip, trpzipalpha has shown a greater tendency to oligomerize in water.     

Conformational effects on tryptophan fluorescence in cyclic hexapeptides

The peptide bond quenches tryptophan fluorescence by excited-state electron transfer, which probably accounts for most of the variation in fluorescence intensity of peptides and proteins. A series of seven peptides was designed with a single tryptophan, identical amino acid composition, and peptide bond as the only known quenching group. The solution structure and side-chain chi(1) rotamer populations of the peptides were determined by one-dimensional and two-dimensional (1)H-NMR. All peptides have a single backbone conformation. The -, psi-angles and chi(1) rotamer populations of tryptophan vary with position in the sequence. The peptides have fluorescence emission maxima of 350-355 nm, quantum yields of 0.04-0.24, and triple exponential fluorescence decays with lifetimes of 4.4-6.6, 1.4-3.2, and 0.2-1.0 ns at 5 degrees C. Lifetimes were correlated with ground-state conformers in six peptides by assigning the major lifetime component to the major NMR-determined chi(1) rotamer. In five peptides the chi(1) = -60 degrees rotamer of tryptophan has lifetimes of 2.7-5.5 ns, depending on local backbone conformation. In one peptide the chi(1) = 180 degrees rotamer has a 0.5-ns lifetime. This series of small peptides vividly demonstrates the dominant role of peptide bond quenching in tryptophan fluorescence.     

Laser photolysis of caged calcium: rates of calcium release by nitrophenyl-EGTA and DM-nitrophen.

Nitrophenyl-EGTA and DM-nitrophen are Ca2+ cages that release Ca2+ when cleaved upon illumination with near-ultraviolet light. Laser photolysis of nitrophenyl-EGTA produced transient intermediates that decayed biexponentially with rates of 500,000 s-1 and 100,000 s-1 in the presence of saturating Ca2+ and 290,000 s-1 and 68,000 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. Laser photolysis of nitrophenyl-EGTA in the presence of Ca2+ and the Ca2+ indicator Ca-orange-5N produced a monotonic increase in the indicator fluorescence, which had a rate of 68,000 s-1 at pH 7.2 and 25 degrees C. Irradiation of DM-nitrophen produced similar results with somewhat slower kinetics. The transient intermediates decayed with rates of 80,000 s-1 and 11,000 s-1 in the presence of Ca2+ and 59,000 s-1 and 3,600 s-1 in the absence of Ca2+ at pH 7.2 and 25 degrees C. The rate of increase in Ca(2+)-indicator fluorescence produced upon photolysis of the DM-nitrophen: Ca2+ complex was 38,000 s-1 at pH 7.2 and 25 degrees C. In contrast, pulses in Ca2+ concentration were generated when the chelator concentrations were more than the total Ca2+ concentration. Photoreleased Ca2+ concentration stabilized under these circumstances to a steady state within 1-2 ms.     

Heat stress induces an aggregation of the light-harvesting complex of photosystem II in spinach plants

Whole spinach (Spinacia oleracea) plants were subjected to heat stress (25 degrees C-50 degrees C) in the dark for 30 min. At temperatures higher than 35 degrees C, CO2 assimilation rate decreased significantly. The maximal efficiency of photosystem II (PSII) photochemistry remained unchanged until 45 degrees C and decreased only slightly at 50 degrees C. Nonphotochemical quenching increased significantly either in the absence or presence of dithiothreitol. There was an appearance of the characteristic band at around 698 nm in 77 K fluorescence emission spectra of leaves. Native green gel of thylakoid membranes isolated immediately from heat-stressed leaves showed that many pigment-protein complexes remained aggregated in the stacking gel. The analyses of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting demonstrated that the aggregates were composed of the main light-harvesting complex of PSII (LHCIIb). To characterize the aggregates, isolated PSII core complexes were incubated at 25 degrees C to 50 degrees C in the dark for 10 min. At temperatures over 35 degrees C, many pigment-protein complexes remained aggregated in the stacking gel of native green gel, and immunoblotting analyses showed that the aggregates were composed of LHCIIb. In addition, isolated LHCII was also incubated at 25 degrees C to 50 degrees C in the dark for 10 min. LHCII remained aggregated in the stacking gel of native green gel at temperatures over 35 degrees C. Massive aggregation of LHCII was clearly observed by using microscope images, which was accompanied by a significant increase in fluorescence quenching. There was a linear relationship between the formation of LHCII aggregates and nonphotochemical quenching in vivo. The results in this study suggest that LHCII aggregates may represent a protective mechanism to dissipate excess excitation energy in heat-stressed plants.     

Transfer of singlet energy within trypsin.

Transfers of singlet energy within trypsin were investigated by measuring the fluorescence absorption anisotropy of its tryptophan residues. A ratio of the anisotropy of trypsin to that for N-acetyl-L-tryptophanamide was determined between 306 and 250 nm. The ratio had an average value of 0.7, whether the trypsin anisotropy was measured at 228 of 296 K. However, trypsin dissolved in 5 M guanidine hydrochloride showed little fluorescence depolarization at 228 K (the anisotropy ratio was approximately equal to 0.9). Thus, there is an extensive conformation-dependent energy transfer between tryptophans in trypsin. The ratio of anisotropies of tyrpsin at 304--270 nm was used to estimate energy transfer from tyrosine to tryptophan. Ratios of 1.8 and 1.7 were obtained at 296 K for the native and guanidinium-unfolded enzyme, respectively. The comparable value for N-acetyl-L-tryptophanamide was 1.7. This indicates that there is little transfer from tyrosine to tryptophan in trypsin at 296 K. As confirmation, the excitation wavelength dependencies of the indole fluorescence quantum yield were the same for native and unfolded trypsin. When experiments were performed at 228 K, the 304--270-nm anisotropy ratios were 2.6 for native and 2.1 for unfolded trypsin at pH2. This indicates that the efficiency of energy transfer from tyrosine to tryptophan increases at low temperatures. A photochemical source of error in the quantitation of the efficiency of energy transfer from tyrosine to tryptophan is also described.     

Chlorophyll-protein composition of the thylakoid membrane from Prochlorothrix hollandica, a prokaryote containing chlorophyll b

The chlorophyll-protein complexes of the thylakoid membrane from Prochlorothrix hollandica were identified following electrophoresis under nondenaturing conditions. Five complexes, CP1-CP5, were resolved and these green bands were analyzed by spectroscopic and immunological methods. CP1 contains the photosystem I (PSI) reaction center, as this complex quenched fluorescence at room temperature, and had a 77 K fluorescence emission peak at 717 nm. CP4 contains the major chlorophyll-a-binding proteins of the photosystem II (PSII) core, because this complex contained polypeptides which cross-reacted to antibodies raised against Chlamydomonas PSII proteins 5 and 6. Furthermore, fluorescence excitation studies at 77 K indicated that only a Chl a is bound to CP4. Complexes CP2, CP3 and CP5 contained functionally bound Chl a and b as judged by absorption spectroscopy at 20 degrees C and fluorescence excitation spectra at 77 K. CP2, CP3 and CP5 all contain polypeptides of 30-33 kDa which are immunologically distinct from the LHC-II complex of higher plant thylakoids.     

Biosynthesis and Characterization of 4-Fluorotryptophan-Labeled Escherichia coli Arginyl-tRNA Synthetase

Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K _m values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of ¹⁹F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.