Fluorescence Characterization of Standard, Mutant and Sweet Corn.

This work resulted in the development of a method based on fluorescence spectroscopy to differentiate between three corn varieties, standard, mutant and sweet, and to characterize the corn variety present in finished products. This was achieved by recording fluorescence emission spectra as a function of excitation wavelength. For a standard, non-transgenic and non-sweet corn, the maximum of the first peak is around 412-414 nm at the excitation wavelength equal to 280 nm and shifts to the longer emission wavelengths as the excitation wavelength increases. Also, the second peak is located at 535 nm or is slightly higher (537 to 540 nm) and does not vary for excitation wavelengths from 280 to 360 nm. For mutant corn, the position of the first peak is located at 420 nm and above for λex = 280 nm, while the second peak starts at 525-530 nm (depending on the mutant) and never reaches 535 nm. Finally, for a sweet corn, the position of the first fluorescence emission peak is around 430 nm. If the sweet corn is non-hybrid, the position of the second emission peak is at 535 nm. A hybrid sweet corn has its second peak around 530 nm. Thus, fluorescence emission at 530 is characteristic of corn that has undergone natural or artificial genetic transformation. Finally, we found simple mathematical equations to calculate the percentage of amylopectin and amylose in a given corn.

Tryptophan 19 residue is the origin of bovine ß-lactoglobulin fluorescence.

ß-Lactoglobulin consists of a single polypeptide of 162 amino acid residues with 2 Trp residues, Trp 19 present in a hydrophobic pocket and Trp 61 present at the surface of the protein near the pocket. This study aimed to characterize the respective contribution of the two Trp residues to the overall fluorescence of the protein. We added for that calcofluor white, an extrinsic fluorophore, which, at high concentration compared to that of the protein, quenches completely emission of hydrophobic Trp residue(s). The study was performed at different pHs by recording fluorescence steady state spectra and measuring fluorescence lifetimes of the Trp-residues using Single Time Photon Counting method. Our results indicate that addition of calcofluor white does not induce a red shift of the tryptophan(s) emission peak (332nm) but only a decrease in the fluorescence intensity. This means that Trp 61 residue does not contribute to the protein emission, tryptophan emission occurs from Trp 19 residue only. Also, excitation spectrum peak position (283nm) of ß-lactoglobulin is not modified upon calcofluor white binding. These results mean that structural rearrangements within ß-lactoglobulin are not occurring upon calcofluor white binding. Energy transfer between Trp 19 residue and calcofluor white occurs with 100% efficiency, i.e. the two fluorophores are very close one to each other (<5Å). This energy transfer is not Forster type. Fluorescence intensity decay of Trp 19 residue occurs with three lifetimes, equal to 0.48, 1.49 and 4.29ns at pH 2 (monomeric state). Very close values were obtained at the different studied pHs (2-12) and where ß-lactoglobulin is at different quaternary structure or present in solution in a mixture of dimers and monomers. Our data are interpreted as the results of emission occurring from different substructures of the tryptophan, reached at the excited state. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the microenvironment of the fluorophore and on the local protein structure.

Relation between proteins tertiary structure, tryptophan fluorescence lifetimes and tryptophan S(o)→(1)L(b) and S(o)→(1)L(a) transitions. Studies on α1-acid glycoprotein and ß-lactoglobulin.

We measured fluorescence lifetimes and fluorescence spectra (excitation and emission) of tryptophan residues of α(1)-acid glycoprotein (three Trp residues) and ß-lactoglobulin (two Trp residues) in absence and presence of 450 µM progesterone. Progesterone binds only to α(1)-acid glycoprotein. In absence of progesterone, each of the two proteins displays three fluorescence lifetimes. Addition of progesterone induces a partial inhibition of the S(o) → (1)L(a) transition without affecting fluorescence lifetimes. The same experiments performed in presence of denatured proteins in 6 M guanidine show that addition of progesterone inhibits partially the S(o) → (1)L(a) transition and its peak is 15 nm shifted to the red compared to that obtained for native proteins. However, the S(o) → (1)L(b) transition position peak is not affected by protein denaturation. Thus, the tertiary structure of the protein plays an important role by modulating the tryptophan electronic transitions. Fluorescence emission decay recorded in absence and presence of progesterone yields three fluorescence lifetimes whether proteins are denatured or not. Thus, protein tertiary structure is not responsible for the presence of three fluorescence lifetimes. These characterize tryptophan substructures reached at the excited states and which population (pre-exponential values) depend on the tryptophan residues interaction with their microenvironment(s) and thus on the global conformation of the protein.

Sub-structures formed in the excited state are responsible for tryptophan residues fluorescence in ß-lactoglobulin.

Origin of tryptophan residues fluorescence in ß-lactoglobulin is analyzed. Fluorescence lifetimes and spectra of ß-lactoglobulin solution are measured at pH going from 2 to 12 and in 6 M guanidine. Tryptophan residues emit with three lifetimes at all conditions. Two lifetimes (0.4-0.5 ns and 2-4 ns) are in the same range of those measured for tryptophan free in solution. Lifetimes in the denatured states are lower than those measured in the native state. Pre-exponential values are modified with the protein structure. Data are identical to those already obtained for other proteins. Fluorescence lifetimes characterize internal states of the tryptophan residues (Tryptophan sub-structures) independently of the tryptophan environments, the third lifetime results from the interaction that is occurring between the Trp residues and its environment. Pre-exponential values characterize substructures populations. In conclusion, tryptophan mission occurs from substates generated in the excited state. This is in good agreement with the theory we described in recent works.

Origin of fluorescence lifetimes in human serum albumin. Studies on native and denatured protein.

Human serum albumin consists of a single polypeptide of 585 amino acid residues with 1 Trp residue. In the present work, we measured fluorescence lifetimes of the protein in both native and denatured states. The results indicate that Trp emission occurs with three lifetimes in both states. Lifetimes values and contribution to the global emission decay differ between the two states. Data are interpreted as the results of an emission occurring from three substructures of the tryptophan formed in the excited state. Two of these substructures are already present for the tryptophan free in solution. The third lifetime is the result of the interaction between the tryptophan residue and surrounding microenvironment. The populations of these substructures characterized by the pre-exponential parameters of the fluorescence lifetimes are dependent on the fluorophore microenvironment and on the global protein structure.

Effect of 1-aminoanthracene (1-AMA) binding on the structure of three lipocalin proteins, the dimeric ß lactoglobulin, the dimeric odorant binding protein and the monomeric α1-acid glycoprotein. Fluorescence spectra and lifetimes studies.

We studied effect of 1-aminoanthracene (1-AMA) binding on the structures of dimeric ß lactoglobulin, dimeric odorant binding protein (OBP) and monomeric α(1)-acid glycoprotein (lipocalin family proteins) by monitoring fluorescence excitation spectra and measuring fluorescence lifetimes of the tryptophan residues of the proteins. Results show that binding of 1-AMA to ß lactoglobulin and OBP modifies their conformation even at low probe concentration compared to that of the proteins. Structural modification induces a red shift of the fluorescence excitation spectra maximum of tryptophan residues accompanied with an increase of the third fluorescence lifetime and a decrease of its pre-exponential factor. These effects were not observed for α(1)-acid glycoprotein, probably as the result of carbohydrate presence. These data raise doubts concerning use of 1-AMA as a probe to study biological properties of ß lactoglobulin and OBP.

Characterization of human serum albumin forms with pH. Fluorescence lifetime studies.

Fluorescence lifetimes of human serum albumin (HSA) tryptophan 214 residue were measured in solution at different pH (from 2 to 12). The results indicate that tryptophan emission occurs with three lifetimes at all pH. However, lifetimes and pre-exponential values are dependent on the pH and thus on the protein form. Three different protein populations have been differentiated: one population for pH 2 and 3 (extended form), the second one from pH 4 to 9 containing HSA migrating (F), normal (N) and basic (B) forms. Another type of population is obvious for pH higher than 9, characterizing the aged (A) form of HSA.

Fluorescence lifetimes of tryptophan: structural origin and relation with So --> 1Lb and So --> 1La transitions.

We measured fluorescence lifetimes of L-Tryptophan dissolved in de-ionized water and in ethanol in the absence and the presence of high progesterone concentrations. The hormone absorbs between 220 and 280 with a peak around 250 nm, while its absorption is equal to zero beyond 280 nm. Tryptophan excitation spectrum recorded in presence of progesterone shows that the S(o) --> 1L(a) transition is completely abolished while the S(o) --> 1L(b) transition is not affected. Emission of L-tryptophan in water occurs with two fluorescence lifetimes, 0.40 and 2.8 ns. In ethanol, three fluorescence lifetimes equal to around 0.2, 1.8 and 4.8 ns were observed. Addition of progesterone to the medium does not affect any of the fluorescence lifetimes indicating clearly that both transitions could induce tryptophan excitation and that recorded fluorescence lifetimes could be assigned to sub-structures generated in the excited state.

Fluorescence spectral resolution of tryptophan residues in bovine and human serum albumins.

Static quenching and time-resolved emission spectra of tryptophan residues of BSA (2 Trp residues) and HSA (1 Trp residue) were performed in the presence of high concentrations of calcofluor white, a fluorophore that is specific to both carbohydrate residues and to hydrophobic sites in proteins. In the absence of calcofluor white, BSA and HSA emit with a maximum at 340 and 330 nm, respectively. Also, tryptophan residues in both proteins fluoresce with three identical lifetimes. Time-resolved spectra of HSA show that the three lifetimes emit at a maximum equal to 330 nm while spectra obtained from BSA show different peak positions for the three lifetimes. At high calcofluor concentrations, steady-state fluorescence emission spectrum of BSA displays a maximum at 330 nm instead of 340 nm in the absence of calcofluor. Fluorescence excitation spectra of the protein recorded in the absence and presence of calcofluor indicate the absence of protein conformational modification upon calcofluor white binding. Time-resolved emission spectra of the three lifetimes show identical peaks equal to 330 nm. Steady-state and time-resolved emission spectra performed on HSA in the presence of calcofluor do not show any modification in the emission peak (330 nm) indicating the absence of any conformational change and confirming the fact that the shift observed for tryptophan residues emission in BSA is the result of fluorescence quenching of Trp-134 residue.

Effect of the secondary structure of carbohydrate residues of alpha 1-acid glycoprotein (orosomucoid) on the local dynamics of Trp residues.

We studied in this work the relation between the secondary structure of the carbohydrate residues of alpha1-acid glycoprotein and the local motions of Trp residues of the protein. We measured for this purpose the fluorescence emission intensity and anisotropy of the Trp residues between -46 and +30 degrees of the sialylated and asialylated protein. Our results indicate that, in both forms, the global profile of the emission intensity with temperature shows that Trp residues display static and collisional interaction with the neighboring amino acids. However, the profile of the asialylated form is more structured than that observed for the sialylated protein. The Y-plot analysis of the emission-anisotropy results indicated that the frictional resistance to rotation of the surface Trp residue is less important in the sialylated protein than in the asialylated form. This result is in good agreement with the fact that, in the asialylated conformation, the carbohydrate residues are closer to the protein surface than in the sialylated form, thereby increasing the contact of the surface Trp residue with the neighboring amino acids. Also, the interaction between the carbohydrate residues and the surface Trp residue contributes to the modification of the frictional resistance to rotation of the fluorophore.

Interaction between carbohydrate residues of alpha(1)-acid glycoprotein (orosomucoid) and progesterone. A fluorescence study.

Interaction between progesterone and the carbohydrate residues of alpha(1)-acid glycoprotein was followed by fluorescence studies using calcofluor white. The fluorophore interacts with polysaccharides and is commonly used in clinical studies. Binding of progesterone to the protein induces a decrease in the fluorescence intensity of calcofluor white, accompanied by a shift to the short wavelengths of its emission maximum. The dissociation constant of the complex was found equal to 8.62 microM. Interaction between progesterone and free calcofluor in solution induces a low decrease in the fluorescence intensity of the fluorophore without any shift of the emission maximum. These results show that in alpha(1)-acid glycoprotein, the binding site of progesterone is very close to the carbohydrate residues. Fluorescence intensity quenching of free calcofluor in solution with cesium ion gives a bimolecular diffusion constant (k(q)) of 2.23 x 10(9) M(-1) s(-1). This value decreases to 0.19 x 10(9) M(-1) s(-1) when calcofluor white is bound to alpha(1)-acid glycoprotein. Binding of progesterone does not modify the value of k(q) of the cesium. Previous studies have shown that the terminal sialic acid residue is mobile, while the other glycannes are rigid [Albani, J. R.; Sillen, A.; Coddeville, B.; Plancke, Y. D.; Engelborghs, Y. Carbohydr. Res. 1999, 322, 87-94]. Red-edge excitation spectra and Perrin plot experiments performed on sialylated and asialylated alpha(1)-acid glycoprotein show that binding of progesterone to alpha(1)-acid glycoprotein does not modify the local dynamics of the carbohydrate residues of the protein.