Effects of pH on benzimidazole fluorescence

Four benzimidazoles (unsubstituted, 5-methyl, 2-ethyl, and 2-ethyl-5-methyl) have been characterized by fluorescence spectroscopy. At low pH (<6), activation at 270 nm caused fluorescence at 305 nm; at high pH (<8), activation at 270 nm caused fluorescence at 365 nm. The relative proportion of peak fluorescence at either 305 or 365 nm was correlated with the pK_a values of the four benzimidazoles. It was concluded that the protonated specie of benzimidazole was fluorescent at 365 nm and the unprotonated specie was also fluorescent at 305 nm.     

A comparison of the intrinsic fluorescence of red kangaroo, horse and sperm whale metmyoglobins

Several metmyoglobins (red kangaroo, horse and sperm whale), containing different numbers of tyrosines, but with invariant tryptophan residues (Trp-7, Trp-14), exhibit intrinsic fluorescence when studied by steady-state front-face fluorometry. The increasing tyrosine content of these myoglobins correlates with a shift in emission maximum to shorter wavelengths with excitation at 280 nm: red kangaroo (Tyr-146) emission maximum 335 nm; horse (Tyr-103, -146) emission maximum 333 nm; sperm whale (Tyr-103, -146, -151) emission maximum 331 nm. Since 280 nm excites both tyrosine and tryptophan, this strongly suggests that tyrosine emission is not completely quenched but also contributes to this fluorescence emission. Upon titration to pH 12.5, there is a reversible shift of the emission maximum to longer wavelengths with an increase greater than 2-fold in fluorescence intensity. With excitation at 305 nm, a tyrosinate-like emission is detected at a pH greater than 12. These studies show that: (1) metmyoglobins, Class B proteins containing both tyrosine and tryptophan residues, exhibit intrinsic fluorescence; (2) tyrosine residues also contribute to the observed steady-state fluorescence emission when excited by light at 280 nm; (3) the ionization of Tyr-146 is likely coupled to protein unfolding.     

A pulse fluorometry study of lipoamide dehydrogenase. Evidence for non-equivalent FAD centers.

The time dependence of the fluorescence of tryptophanyl and flavin residues in lipoamide dehydrogenase has been investigated with single-photon decay spectroscopy. When the two FAD molecules in the enzyme were directly excited the decay could only be analyzed in a sum of two exponentials with equal amplitudes. This phenomenon was observed at 4 degrees C (tau-1 = 0.8 ns, tau-2 = 4.7 ns) and at 20 degrees C (tau-1 = 0.8 ns, tau-2 = 3.4 ns) irrespective of the emission and excitation wavelengths. This result reveals a difference in the nature of the two FAD centers. By excitation at 290 nm the fluorescence decay curves of tryptophan and FAD were obtained. The decays are analyzed in terms of energy transfer from tryptophanyl to flavin residues. The results, which are in good agreement with those obtained previously with static fluorescence methods, show that one of the two tryptophanyl residues within the subunit transfers its excitation energy to the flavin located at a distance of 1.5 nm.     

Tyrosine and tyrosinate fluorescence of S-100b. A time-resolved nanosecond fluorescence study. The effect of pH, Ca(II), and Zn(II)

The properties of the tyrosine and tyrosinate emissions from brain S-100b have been studied by nanosecond time-resolved fluorescence at emission wavelengths in the range 305 to 365 nm. The effect of pH on the fluorescence has been studied at pH 6.5, 7.5, and 8.5 for the Ca(II) apo and holo forms of the protein, and for the apo and holo forms in the presence and absence of Zn(II) at pH 7.5. The fluorescence decay is biexponential at pH 8.5 and triexponential at pH 6.5 and 7.5. The three components of the decay have wavelength and metal ion dependent lifetimes in the ranges 0.06 to 1.05 ns, 0.49 to 3.76 ns, and 3.60 to 14.5 ns. The observation of a long lifetime component at wavelengths characteristic of emission from tyrosinate suggests that in class A proteins this may be a useful diagnostic of the environment of tyrosine in their native structures. The time-resolved emission spectra provide evidence for efficient, subnanosecond protolysis of the excited state of the single tyrosine (Tyr17) under all conditions studied except in 6 M guanidium chloride in which the protein shows only emission from tyrosine (lambda em 305 nm), suggesting that the tyrosinate emission is a property of the tertiary structure of the native protein. The Zn(II)-dependence of the fluorescence is fully consistent with the earlier suggestion that Tyr17 is near the Zn(II) binding site and remote from the high affinity Ca(II) binding site.     

In Situ pH Measurement in Partly Frozen Aqueous Solution Using the Fluorescent Probe 8-Hydroxypyrene-1,3,6-Trisulfonic Acid

A method based on the fluorescence probe 8-hydroxypyrene-1,3,6-trisulfonic acid for in situ measurement of pH in partly frozen aqueous solutions was developed using multifrequency, phase-modulated fluorescence spectroscopy inherently correcting for light scattering. The probe was determined to have pK _a = 7.72 ± 0.03 at 25.0 °C extrapolated to zero ionic strength with as derived from temperature dependence (5 to 25 °C investigated). Ionic strength dependence of pK _a determined experimentally was described using Debye–Hückel formalism for ionic strength up to 3 M. Temperature and ionic strength dependence were combined to yield for determination of pH at subzero temperatures with α experimentally determined from the ratio between fluorescence intensity after excitation at 454 and 415 nm, α = FI(454 nm)/2.5·FI(415 nm). Fluorescence could be described as a decay of a single excited state with a fluorescence life time of 5.40 ± 0.05 ns at 25 °C, and excited state acid–base equilibration was shown not to interfere with the pH measurement. Using the method, pH of a 0.25 M phosphate buffer with pH = 6.8 at 25 °C was shown to decrease gradually to pH = 4.2 in the ice slurry at −13 °C.     

Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.     

X-ray diffraction and electron microscopy of a light meromyosin tactoid

The structure of a tactoid of light meromyosin with a 43-nm periodicity was studied by both X-ray diffraction and electron microscopy. Such tactoids were formed from light meromyosin prepared by a short tryptic digestion (5 min) of myosin.A strong magnetic field (6 kgauss) was employed to obtain oriented specimens of tactoids for X-ray diffraction. The oriented tactoids gave equatorial reflections from a rectangular lattice with a unit cell of 6·5 nm × 3·9 nm (at pH 6·6) in a plane perpendicular to the long axis of the tactoid. This lattice shrank anisotropically when the pH was lowered. The meridional reflections could be indexed as orders of 42·93 ± 0·05 nm.The tactoids were frequently associated with sheet-like structures termed banded sheets. In negative stain these banded sheets showed the same band pattern as the tactoids with 10 nm wide light and 33 nm wide dark bands. However, in thin banded sheets the density of neighbouring dark bands alternated so that the true axial repeat was 86 nm. Optical diffraction showed that the face-on view of the banded sheet had a unit cell of 3·6 nm × 86 nm.From these observations a plausible model for the structure of the light meromyosin tactoid has been deduced. In this model the tactoid is made by a stacking of unit layers. A unit cell (6·5 nm × 3·9 nm × 86 nm) contains four light meromyosin molecules, each 90 nm long and packed co-planar, not all of which are in an identical environment. The molecules make parallel interactions with staggers of 86 and 43 nm and antiparallel interactions with overlaps of 84 and 41 nm.     

Use of the green fluorescent protein and its mutants in quantitative fluorescence microscopy.

We have investigated properties relevant to quantitative imaging in living cells of five green fluorescent protein (GFP) variants that have been used extensively or are potentially useful. We measured the extinction coefficients, quantum yields, pH effects, photobleaching effects, and temperature-dependent chromophore formation of wtGFP, alphaGFP (F99S/M153T/V163A), S65T, EGFP (F64L/S65T), and a blue-shifted variant, EBFP (F64L/S65T/Y66H/Y145F). Absorbance and fluorescence spectroscopy showed little difference between the extinction coefficients and quantum yields of wtGFP and alphaGFP. In contrast, S65T and EGFP extinction coefficients made them both approximately 6-fold brighter than wtGFP when excited at 488 nm, and EBFP absorbed more strongly than the wtGFP when excited in the near-UV wavelength region, although it had a much lower quantum efficiency. When excited at 488 nm, the GFPs were all more resistant to photobleaching than fluorescein. However, the wtGFP and alphaGFP photobleaching patterns showed initial increases in fluorescence emission caused by photoconversion of the protein chromophore. The wtGFP fluorescence decreased more quickly when excited at 395 nm than 488 nm, but it was still more photostable than the EBFP when excited at this wavelength. The wtGFP and alphaGFP were quite stable over a broad pH range, but fluorescence of the other variants decreased rapidly below pH 7. When expressed in bacteria, chromophore formation in wtGFP and S65T was found to be less efficient at 37 degrees C than at 28 degrees C, but the other three variants showed little differences between 37 degrees C and 28 degrees C. In conclusion, no single GFP variant is ideal for every application, but each one offers advantages and disadvantages for quantitative imaging in living cells.     

Effects of an interchain disulfide bond on tropomyosin structure: intrinsic fluorescence and circular dichroism studies

An interchain disulfide crosslink was introduced into rabbit skeletal tropomyosin (TM) at Cys190 by two different methods under non-denaturing conditions. The effects of the crosslink on the structure of tropomyosin were investigated by fluorescence and circular dichroism methods as a function of temperature and guanidine · hydrochloride concentration. Four different preparations were studied: Nbs₂-TM, red-TM crosslinked with Ellman's reagent, 5,5′-dithiobis(2-nitrobenzoate); O₂-TM, TM whose SH groups were air-oxidized; red-TM, TM reduced with dithiothreitol; IA-TM, red-TM whose SH groups were blocked with iodoacetamide. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis studies indicated that SS crosslinks were quantitatively introduced between the subunits of TM for Nbs₂-TM and O₂-TM. In the completely folded state (below 25 °C or in the absence of denaturant) and in the unfolded state (above 65 °C or greater than 4 m-guanidine · hydrochloride) all of the samples had the same Tyr fluorescence quantum yield, accessibility to acrylamide fluorescence quenching, fluorescence polarization and mean residue rotation at 222 nm. Thermal and denaturant-induced unfolding profiles at pH 7.5 were obtained for each sample with measurements of these parameters. The main transition at about 45 °C or 2 m-guanidine · hydrochloride was shifted about +7 deg. C and 0.8 m in guanidine · hydrochloride, respectively, for the crosslinked samples as compared to the uncrosslinked samples. In addition, a destabilizing pretransition was observed in the 30 to 45 °C region or the 0 to 2 m-guanidine · hydrochloride region only for the crosslinked samples when polarization or ellipticity was measured. Studies of the ability of Nbs₂ to crosslink red-TM as a function of guanidine · hydrochloride concentration indicated that the chains separate at Cys190 between 0 and 2 m-guanidine · hydrochloride before they dissociate. Thus, the effect of the SS crosslink at Cys190 on the conformation of TM at physiological temperatures appears to be related to the inherent instability of the molecule in this region of the sequence.     

A fluorescence study of egg white riboflavin-binding protein

1. Denaturation of riboflavin-binding protein (RBP) by guanidine hydrochloride (Gu-HCl) was investigated by measruing the fluorescence of the protein. The denaturation-renaturation processes of RBP by Gu-HCl were fully reversible. The apo-RBP fluorescence had an emission maximum at 343 nm in the absence of Gu-HCl, and at 350 nm in the presence of 4M Gu-HCl, which completely denatured the protein. The relative fluorescence yield of apo-RBP in the presence of 4 M Gu-HCl was about 170% of that in the absence of Gu-HCl. The affinity of native apo-RBP for riboflavin was very strong, while riboflavin was not bound to the denatured form. The equilibrium system of apo-RBP and riboflavin in solutions containing Gu-HCl at various concentrations was analyzed by measuring riboflavin fluorescence. 2. The quenching of apo-RBP fluorescence, probably the fluorescence of tryptophanyl residues, by iodide anions and cesium cations was measured. The fluorescence of apo-RBP in the presence of 4 M Gu-HCl was quenched considerably by iodide and cesium, and Stern-Volmer plots were linear. However, the fluorescence of native apo-RBP was scarcely quenched by iodide or cesium. This suggested that tryptophanyl residues buried inside apo-RBP were responsible for most of the tryptophanyl fluorescence of native apo-RBP.     

Effect of bovine growth hormone on rat liver plasma membranes as studied by circular dichroism and fluorescence using the extrinsic probe 7,12-dimethylbenzanthracene

Conformational changes produced by in vitro bovine growth hormone addition to plasma membranes of hypophysectomized rat liver proteins and lipids have been studied by circular dichroism as well as intrinsic and extrinsic fluorescence. 7,12-Dimethylbenzanthracene has been used as a fluorescent probe of changes in membrane structure. The exposure of membranes to bovine growth hormone produced a change in membrane negative ellipticity. Dimethylbenzanthracene at concentrations similar to those employed in fluorescence studies had no effect on the membrane circular dichroism spectrum. Its presence did, however, prevent a response to growth hormone. There was a decrease in peak fluorescence intensity and a peak shift when bovine growth hormone (0.5 · 10^?12 M) was added to liver membranes. The addition of dimethylbenzanthracene (1.6 · 10^?6 M) to membranes resulted in a decrease in the intensity of the protein fluorescence peak at 335 nm and the appearance of two peaks at 430 and 407 nm, assignable to the probe. The addition of bovine growth hormone (0.5 · 10^?12 M) produced a decrease in fluorescence at 335 nm and also in the peaks at 407 and 430 nm. These data are consistent with the conclusion that bovine growth hormone produces a conformational change in rat liver plasma membrane proteins and lipids.     

Interaction of the serum amyloid A proteins with phospholipid

The serum amyloid A proteins (SAA) are transported in plasma in association with the high density lipoproteins. We have studied the solution properties of two of the polymorphic forms of SAA, SAA1 and SAA4, and compared the lipid-binding properties of SAA4 to those of the well characterized apolipoproteins, apo-A-I, apo-A-II, and apo-C-III. SAA4 was monomeric at pH 2.9 but considerable self-association was demonstrated at pH 8.2, even in the presence of 1.0 M guanidine HCl. SAA4 differed from the apolipoproteins in its ability to disrupt multilamellar dimyristoylphosphatidylcholine (DMPC) liposomes and generate bilayer discs. Apo-A-I, apo-A-II, and apo-C-III reduced the turbidity of DMPC dispersions at protein:lipid molar ratios of 1:200. SAA4, however, increased turbidity at molar ratios of 1:250 and 1:100 even when preincubated in guanidine HCl before addition to liposomes. Optical density decreased only at ratios of 1:50 and 1:25. At an SAA4:DMPC ratio of 1:50, discoidal particles (long axis, 28.1 nm; short axis, 4.4 nm) were formed which were similar to those produced by apo-C-III. Lipid binding induced changes in SAA4 conformation similar to those observed in the apolipoproteins. The alpha-helical content and intrinsic tryptophanyl fluorescence were increased and quenching of tryptophanyl fluorescence by acrylamide was reduced in the presence of DMPC. In addition, SAA4 as well as the apolipoproteins broadened the range and increased the temperature of the gel-liquid crystal transition temperature of DMPC.     

The Spectral Properties of (-)-Epigallocatechin 3-O-Gallate (EGCG) Fluorescence in Different Solvents: Dependence on Solvent Polarity

(-)-Epigallocatechin 3-O-gallate (EGCG) a molecule found in green tea and known for a plethora of bioactive properties is an inhibitor of heat shock protein 90 (HSP90), a protein of interest as a target for cancer and neuroprotection. Determination of the spectral properties of EGCG fluorescence in environments similar to those of binding sites found in proteins provides an important tool to directly study protein-EGCG interactions. The goal of this study is to examine the spectral properties of EGCG fluorescence in an aqueous buffer (AB) at pH=7.0, acetonitrile (AN) (a polar aprotic solvent), dimethylsulfoxide (DMSO) (a polar aprotic solvent), and ethanol (EtOH) (a polar protic solvent). We demonstrate that EGCG is a highly fluorescent molecule when excited at approximately 275 nm with emission maxima between 350 and 400 nm depending on solvent. Another smaller excitation peak was found when EGCG is excited at approximately 235 nm with maximum emission between 340 and 400 nm. We found that the fluorescence intensity (FI) of EGCG in AB at pH=7.0 is significantly quenched, and that it is about 85 times higher in an aprotic solvent DMSO. The Stokes shifts of EGCG fluorescence were determined by solvent polarity. In addition, while the emission maxima of EGCG fluorescence in AB, DMSO, and EtOH follow the Lippert-Mataga equation, its fluorescence in AN points to non-specific solvent effects on EGCG fluorescence. We conclude that significant solvent-dependent changes in both fluorescence intensity and fluorescence emission shifts can be effectively used to distinguish EGCG in aqueous solutions from EGCG in environments of different polarity, and, thus, can be used to study specific EGCG binding to protein binding sites where the environment is often different from aqueous in terms of polarity.     

Determination of the degree of acetylation of chitosans by first derivative ultraviolet spectrophotometry

The degree of acetylation of chitosan can be determined in acetic acid solutions (～0·01m) containing 1 g dry chitosan per litre by first derivative ultraviolet spectrophotometry at 199 nm. At this wavelength, the N-acetylglucosamine absorbance readings are linearly dependent on concentration and are not influenced by the presence of acetic acid. Correction factors for the contribution of glucosamine in highly deacetylated chitosans can be easily derived. Typical results for the chitosan of Euphausia superba are: degree of acetylation, 42·6; relative standard deviation, 1·3%; confidence limits, ±0·7. This method is simpler, more precise and faster than the infrared method. Sonication of chitosan solutions leads to immediate chain degradation and to detectable deacetylation after more prolonged periods of time, especially when the pH is 1·0.     

Use of fluorescence enhancement technique to study bilirubin–albumin interaction

Bilirubin–albumin solution gave an emission spectrum in the wavelength range 500–600 nm with emission maxima at 528 nm when excited at 487 nm. The magnitude of fluorescence intensity increased on increasing bilirubin/albumin molar ratio. At three different albumin concentrations, namely, 1.0, 2.5 and 10.0 μM, there was an initial linear increase in fluorescence up to a molar ratio 1.0 in all cases beyond which it sloped off or decreased. This fluorescence enhancement was used to calculate the binding parameters of bilirubin–albumin interaction and the value of binding constant was found to be 1.72×10⁷ l/mol similar to the published values obtained with other methods. Different serum albumins, namely, human (HSA), goat (GSA), pig (PSA) and dog serum albumins (DSA) bound bilirubin with almost the same affinity when studied by the technique of fluorescence enhancement. Bilirubin–albumin interaction was also studied at different pH and ionic strengths. There was a decrease in bilirubin–albumin complex formation on either decreasing the pH from 9.0 to 7.0 or increasing the ionic strength from 0.15 to 1.0. These results suggest that the technique of fluorescence enhancement can be used successfully to study the bilirubin–albumin interaction.     

Photoconversion of DAPI and Hoechst dyes to green and red-emitting forms after exposure to UV excitation

The fluorescent dye 4′-6-Diamidino-2-phenylindole (DAPI) is frequently used in fluorescence microscopy as a chromosome and nuclear stain because of its high specificity for DNA. Normally, DAPI bound to DNA is maximally excited by ultraviolet (UV) light at 358 nm, and emits maximally in the blue range, at 461 nm. Hoechst dyes 33258 and 33342 have similar excitation and emission spectra and are also used to stain nuclei and chromosomes. It has been reported that exposure to UV can convert DAPI and Hoechst dyes to forms that are excited by blue light and emit green fluorescence, potentially confusing the interpretation of experiments that use more than one fluorochrome. The work reported here shows that these dyes can also be converted to forms that are excited by green light and emit red fluorescence. This was observed both in whole tissues and in mitotic chromosome spreads, and could be seen with less than 10-s exposure to UV. In most cases, the red form of fluorescence was more intense than the green form. Therefore, appropriate care should be exercised when examining tissues, capturing images, or interpreting images in experiments that use these dyes in combination with other fluorochromes.     

The effect of evolution on homologous proteins: a comparison between the chromophore microenvironments of Italian water buffalo (Bos bubalus, L.) and sperm whale apomyoglobin.

The perturbing effect of guanidium hydrochloride and pH on the molecular structure of water buffalo apomyoglobin has been investigated by circular dichroism in the far and near ultraviolet and by fluorescence. In the wavelength region between 320 and 260 nm the circular dichroic spectrum of the globin is highly structured and the contributions of the aromatic chromophores have been resolved. Buffalo apomyoglobin undergoes a structural transition at neutral pH which involves elements of the secondary and tertiary structure, as indicated by changes of dichroic activity of the peptide and aromatic chromophores and the fluorescence of the two tryptophanyl residues. The possibility of charge-transfer complex between indole and imidazole is discussed. A major structural transition with abrupt unfolding takes place in the pH region between 5.6 and 4.3. Below pH 4.3 the peptide helical residues, which survive the acid transition, appear to be resistent to further acidification to pH 2.0 while tryptophanyl emission is quenched and shifted to longer wavelengths. A structural transition occurs also in alkali above pH 10, which has been detected by the same techniques. The relationships between buffalo and sperm whale apomyoglobin are discussed.     

Subunit interactions in rabbit-muscle glyceraldehyde-phosphate dehydrogenase, as measured by NAD+ and NADH binding.

1. The binding parameters for NADH and NAD+ to rabbit-muscle glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12) have been measured by quenching of the flourescence of the protein and the NADH. 2. The fact that the degree of protein fluorescence quenching by bound NAD+ or NADH, excited at 285 nm and measured at 340 nm ('blue' tryptophans), is not linearly related to the saturation functions of these nucleotides, leads to a slight overestimation of the interaction energy and an underestimation of the concentration of sites, if linearity is assumed. 3. This is also the case for NADH, but not for NAD+, when the protein fluorescence is excited at 305 nm and measured at 390 nm ('red' tryptophans). 4. The binding of NAD+ can be described by a model in which the binding of NAD+, via negative interactions within the dimer, induces weaker binding sites, with the result that the microscopic dissociation constant is 0.08 microM at low saturation and 0.18 microM for the holoenzyme. 5. The binding of NADH can be described on the basis of the same model, the dissociation constant at low saturation being 0.5 microM and of the holoenzyme 1.0 microM. 6. The fluorescence of bound NADH is not sensitive to the conformational changes that cause the decrease in affinity of bound NAD+ or NADH. 7. The binding of NAD+ to the 3-phosphoglyceroyl enzyme can be described by a dissociation constant that is at least two orders of magnitude greater than the dissociation constants of the unacylated enzyme. The affinity of NAD+ to this form of the enzyme is in agreement with the Ki calculated from product inhibition by NAD+ of the reductive dephosphorylation of 1,3-diphosphoglycerate.     

Biosorption of iron(III)–cyanide complex anions to Rhizopus arrhizus: application of adsorption isotherms

The biosorption of iron(III)–cyanide complex anions to Rhizopus arrhizus was investigated. The iron(III)–cyanide complex ion binding capacity of the biosorbent was a function of initial pH, initial iron(III)–cyanide complex ion and biosorbent concentration. These results indicated that a significant reduction of iron(III)–cyanide complex ions was achieved at pH 13, a highly alkaline condition. The maximum loading capacity of biosorbent was 612·2 mg g⁻¹ at 1996·2 mg litre⁻¹ initial iron(III)–cyanide complex ion concentration at this pH. The Freundlich, Langmuir and Redlich–Peterson adsorption models were fitted to the equilibrium data at pH 3·0, 7·0 and 13·0. The equilibrium data could be best fitted to by all the adsorption models over the entire concentration range (50–2000 mg litre⁻¹) at pH 13.     

Absorption and fluorescence spectra ofS-quinolylethylated Kunitz soybean trypsin inhibitor

Disulfide bonds in soybean trypsin inhibitor (Kunitz) were simultaneously reduced and alkylated using tri-n-butylphosphine and 2-vinylquinoline at pH 7.6 in 0.11 M Tris-4.4 M urea, 41% ethanol. The resulting S-β-2-quinolylethylated protein (2-QE-STI) has a new absorption peak at 315–318 nm. Its quinoline fluorescence can be excited above 310 nm independently of intrinsic protein fluorescence. Free 2-quinolylethylcysteine (2-QEC) shows unexpectedly weak fluorescence. Quinoline absorption in 2-QEC and 2-QE-STI changes with pH. The apparentpK values determined spectrophotometrically are near 5 for 2-QEC and 3 for 2-QE-STI. Fluorescence decreased with increasing pH and in the presence of chloride ions. Both structural and charge effects thus appear to influence the absorption and fluorescence of the quinoline group. Corrected fluorescence emission (excited at 316 nm) of neutral 2-QE-STI diluted in 0.1 N H₂SO₄ was directly proportional to concentration in the range 0.4–8 μm 2-QEC. The 2-QEC content of the protein derivative determined by UV absorption at pH 1.5 was in agreement with the expected value of four residues per mole. Fluorescence measurements ofS-2-quinolylethylated proteins may be especially useful as a sensitive, specific assay for cyst(e)ine residues.