Tryptophan In Solution Research Articles

Impact of Methionine and Tryptophan Supplementation on Silk Gland Weight and Tissue Somatic Index in Eri Silkworm (Samia ricini Donovan)

This study evaluated the impact of methionine and tryptophan supplementation on the silk gland weight and Silk Gland Tissue Somatic Index (SGTSI) of eri silkworm, Samia ricini Donovan, a key species in vanya silk production. Freshly collected castor leaves were fortified with methionine and tryptophan solutions at various concentrations and fed to silkworm larvae from the third instar until maturity. The treatments significantly enhanced silk gland weight and SGTSI compared to the control. The highest silk gland weight (1.52g) and SGTSI (25.16%) were observed in larvae fed with tryptophan at 500 ppm (T6), followed by methionine at 500 ppm (T3) with 1.47g and 24.61%, respectively. Control larvae exhibited the lowest values (1.07g and 19.43%). Combination treatments of methionine and tryptophan showed less improvement compared to individual treatments. These findings suggest that dietary supplementation with specific amino acids could enhance silk gland development and silk production.

Improving monitoring of dissolved organic matter from the wastewater treatment plant to the receiving environment: A new high-frequency in situ fluorescence sensor capable of analyzing 29 pairs of Ex/Em wavelengths

A high-frequency, in situ fluorescence probe, called Fluocopée®, has been developed in order to better monitor variations in both the quality and quantity of dissolved organic matter within various aquatic environments (e.g. wastewater, receiving environments) thanks to a wide choice of 29 measured Excitation/Emission wavelength pairs. This advance pave the way to new measurement possibilities in comparison with existing probes, which are usually only able to measure 1–4 fluorophores. The qualification tests of the Fluocopée® probe indicate a high level of accuracy for the measurements of tyrosine, tryptophan and humic acids solutions. Good repeatability and reproducibility are also observed. For the first time, this tool has been deployed in an urban watershed (Bougival, Seine River, downstream of Paris) and in the settled effluent from a wastewater treatment plant (Seine aval, Achères, France). This new high-frequency in situ probe offers great application potential, including organic matter quality and quantity monitoring at drinking and wastewater treatment plants (treatment optimization) and in continental and marine waters (the fate of organic matter in biogeochemical cycles).

Obtaining of enriched di-tryptophan fractions by gamma irradiation of free tryptophan solutions

Obtaining of enriched di-tryptophan fractions by gamma irradiation of free tryptophan solutions

Comparative proteomic analysis of two contrasting cultivars reveals the mechanism of sugar beet response to freezing stress

Sugar beet (Beta vulgaris L.) is an economic crop integrating grain and feed production, and is a major source of sucrose. Freezing stress can cause dehydration and necrosis of seedlings. Therefore, it is important to study the basic physiological and proteomic mechanisms of different sugar beet cultivars under freezing stress. In this study, compared with freezing-sensitive (SME) cultivar, the freezing-tolerant (TBP) cultivar showed higher photosynthetic rate, antioxidant enzyme activity, proline and abscisic acid levels, lower malondialdehyde and gibberellin levels and relative conductivity under freezing stress. Moreover, comparative proteomic analysis revealed that proteins mapped to photosynthesis, tryptophan synthesis, anthocyanin synthesis, tricarboxylic acid cycle, reactive oxygen species scavenging and sucrose metabolism pathways were more abundant in TBP, indicating that these metabolic pathways may be beneficial to improve freezing tolerance. In particular, compared with SME cultivar, proteins involved in tryptophan synthesis were found to be upregulated only in TBP cultivar under freezing stress, including phospho-2-dehydro-3-deoxyheptonate aldolase 1, probable inactive shikimate kinase like 1, and anthranilate synthase alpha subunit 2. In addition, quantitative real-time polymerase chain reaction analysis showed that mRNA expression of these three proteins was significantly upregulated in the freezing stress (−5 ℃) vs. control group in the TBP cultivar. Additionally, tryptophan content in TBP leaves increased significantly under freezing stress, while in SME it showed no significant change. Furthermore, the application of exogenous tryptophan solution significantly reduced the mortality rate of sugar beet seedlings (17-day-old) under freezing stress. This study provides a foundation for further functional analysis to fully understand the molecular mechanisms of adaptation to freezing stress in sugar beet.

Ultraviolet Resonance Raman Spectra of Serum Albumins.

The ultraviolet resonance Raman (UVRR) spectra of the two proteins bovine serum albumin (BSA) and human serum albumin (HSA) in an aqueous solution are compared with the aim to distinguish between them based on their very similar amino acid composition and structure and to obtain signals from tryptophan that has only very few residues. Comparison of the protein spectra with solutions of tryptophan, tyrosine, and phenylalanine in comparative ratios as in the two proteins shows that at an excitation wavelength of 220 nm, the spectra are dominated by the strong resonant contribution from these three amino acids. While the strong enhancement of two and one single tryptophan residue in BSA and HSA, respectively, results in pronounced bands assigned to fundamental vibrations of tryptophan, its weaker overtones and combination bands do not play a major role in the spectral range above 1800 cm-1. There, the protein spectra clearly reveal the signals of overtones and combination bands of phenylalanine and tyrosine. Assignments of spectral features in the range of Raman shifts from 3800 to 5100 cm-1 to combinations comprising fundamentals and overtones of tyrosine were supported by spectra of amino acid mixtures that contain deuterated tyrosine. The information in the high-frequency region of the UVRR spectra could provide information that is complementary to near-infrared absorption spectroscopy of the proteins.

Streamlined Efficient Synthesis and Antioxidant Activity of γ-[Glutamyl](n≥1)-tryptophan Peptides by Glutaminase from Bacillus amyloliquefaciens.

As a group of naturally occurring peptides in various foods, γ-glutamyl peptides possess a unique Kokumi taste and health benefits. However, few studies have focused on the functionality of γ-glutamyl peptides. In this study, the γ-[glutamyl] (n=1, 2, 3)-tryptophan peptides were synthesized from a solution of glutamine (Gln) and tryptophan (Trp) employing L-glutaminase from Bacillus amyloliquefaciens. Four different γ-glutamyl peptides were identified from the reaction mixture by UPLC-Q-TOF-MS/MS. Under optimal conditions of pH 10, 37 °C, 3 h, 0.1 mol/L Gln: 0.1 mol/L Trp = 1:3, and glutaminase at 0.1% (m/v), the yields of γ-l-glutamyl-l-tryptophan (γ-EW), γ-l-glutamyl-γ-l-glutamyl-l-tryptophan (γ-EEW) and γ-l-glutamyl-γ-l-glutamyl-γ-l-glutamyl-l-tryptophan (γ-EEEW) were 51.02%, 26.12% and 1.91% respectively. The antioxidant properties of the reaction mixture and the two peptides (γ-EW, γ-EEW) identified from the reaction media were further compared. Results showed that γ-EW exhibited the highest DPPH•, ABTS•+ and O2•--scavenging activity (EC50 = 0.2999 mg/mL, 67.6597 μg/mL and 5.99 mg/mL, respectively) and reducing power (EC50 = 4.61 mg/mL), while γ-EEW demonstrated the highest iron-chelating activity (76.22%). Thus, the synthesized mixture may be used as a potential source of antioxidant peptides for food and nutraceutical applications.

Comparison of spectral and temporal fluorescence parameters of aqueous tryptophan solutions frozen in the light and in the dark

Comparative investigation of temperature dependences of the fast and slow fluorescence decay components in an aqueous solution of tryptophan molecules, frozen in the light and in the dark, was made in the range of –170 °C to + 20 °C. A quantitative analysis of the model of direct and reverse electronic transitions in tryptophan molecule from an excited state to the ground state and to the charge transfer state (CTS) was carried out. Three main spectral regions of tryptophan fluorescence are distinguished that differ in behavior of temperature dependences of the transition rates from the excited state of tryptophan to CTS. The key role of the hydrogen bond system dynamics determining the nonlinear behavior of tryptophan fluorescence parameters in the selected spectral regions is shown. The nonlinear behavior of the fluorescence lifetime, position and shape of fluorescence spectra with changing temperature is determined by tryptophan interaction with an aqueous medium. Besides, the rate of solvation shift of fluorescence spectra is observed. The important role of temperature rearrangements in the system of hydrogen bonds of the water solution surrounding the tryptophan molecule in the excited state is shown.The obtained results can be used to explain the key role of hydrogen bonds in the processes of charge transfer and energy migration in biological systems.

Current-Voltage and Transport Characteristics of Heterogeneous Ion-Exchange Membranes in Electrodialysis of Solutions Containing a Heterocyclic Amino Acid and a Strong Electrolyte.

The alterations in current-voltage and transport characteristics of highly basic and strongly acidic ion-exchange membranes, during the electrodialysis of solutions containing a heterocyclic amino acid and a strong electrolyte, were studied. An increase in the catalytic activity of the water splitting process at the surface of heterogeneous MK-40 and MA-41 membranes upon prolonged contact with proline and tryptophan solutions was found. A significant effect of electroconvection on the components mass transfer through the cation-exchange membrane in the intensive current mode of electrodialysis was revealed for the solution containing a heterocyclic amino acid along with mineral salt (NaCl). This led to a reduction in the length of the "plateau" of the membrane's current-voltage characteristics, in comparison with the characteristics for an individual sodium chloride solution with the same concentration. The changes in the characteristics of the studied ion-exchange membranes caused by contact with solutions containing heterocyclic amino acids during electrodialysis were reversible when applying electrochemical regeneration (cleaning in place) using the overlimiting current mode, corresponding to the region of facilitated transport for these ampholytes.

A new fluorescence-based methodology for studying bioaerosol scavenging processes using a hyperspectral LIF-LIDAR remote sensing system

This paper presents a novel experimental approach to in-situ study of atmospheric phenomena such as nucleation scavenging by biological seeds, bio-droplet dehydration, and bioaerosol's particle scavenging by raindrops. Our methodology is based on the analysis of the dynamical changes of fluorescence signal. We use a remote sensing system based on a homebuilt hyperspectral laser induced fluorescence (LIF) Lidar to measure the transient back-fluorescence and backscattering signals. The spectral line shape of the transient fluorescence associated with an aerosolized tryptophan solution was first analyzed in the laboratory. It then used to study bioaerosol phase transitions between wet and dry conditions. The experiments were first conducted in a dynamic aerosol cell where we repetitively create and monitor the droplets containing bioaerosol cloud starting from its early formation till its total evaporation. The LIF-Lidar was used to simultaneously measure back-fluorescence, scattering and transmission. These measurements were synchronized with the generation of droplets containing bioaerosol and with the monitoring of aerosol's size distribution and ambient conditions. A novel optical receiver design was used to simultaneously detect both back-fluorescence polarization components. Results showed that along with droplet's evaporation process, bioaerosol's fluorescence spectrum exhibit a blue shift, known as the dynamic Stokes-shifts, of ∼2000 cm−1 and an increase in its fluorescence anisotropy. To the best of our knowledge, this is the first report of fluorescence Stokes-shifts and anisotropy within microdroplets containing a biological solution due to wet-dry phase transitions. This method was also used to quantify scavenging of biological particle by raindrops from 100 m. It shows that valuable information can be derived from analyzing the fluorescence spectrum of bioaerosol within a cloud and demonstrate the potential of a LIF-LIDAR remote system to perform in-situ studies of scavenging processes.

Explicit Projection of Stokes Shifts onto Solvent Motion in an Aqueous Liquid and Linear Response Theory.

We investigate the molecular origin of the fluorescence Stokes shift in an aqueous liquid. By examining the speed of energy change, the solvation response function is explicitly projected onto the translational and rotational motions of water molecules for both nonequilibrium relaxation and equilibrium fluctuations. Molecular dynamics simulations of a tryptophan solution show that these two processes have highly consistent dynamics, not only for the total response function but also for the decomposed components in terms of specific molecular movements. We found that the rotational mode governs the relaxation of the Stokes shift, whereas the translational mode contributes non-negligibly with slower dynamics. This consistency implies the similarity of the underlying translational and rotational movements of water molecules as the system is far away from and at equilibrium, supporting the validity of the linear response theory at the molecular level. The decomposition methodology is also applicable to a rigid solvent.

A Thermodynamic Model for Interpreting Tryptophan Excitation-Energy-Dependent Fluorescence Spectra Provides Insight Into Protein Conformational Sampling and Stability.

It is now over 30 years since Demchenko and Ladokhin first posited the potential of the tryptophan red edge excitation shift (REES) effect to capture information on protein molecular dynamics. While there have been many key efforts in the intervening years, a biophysical thermodynamic model to quantify the relationship between the REES effect and protein flexibility has been lacking. Without such a model the full potential of the REES effect cannot be realized. Here, we present a thermodynamic model of the tryptophan REES effect that captures information on protein conformational flexibility, even with proteins containing multiple tryptophan residues. Our study incorporates exemplars at every scale, from tryptophan in solution, single tryptophan peptides, to multitryptophan proteins, with examples including a structurally disordered peptide, de novo designed enzyme, human regulatory protein, therapeutic monoclonal antibodies in active commercial development, and a mesophilic and hyperthermophilic enzyme. Combined, our model and data suggest a route forward for the experimental measurement of the protein REES effect and point to the potential for integrating biomolecular simulation with experimental data to yield novel insights.

Chirality and concentration detection of biomolecules based on spin Hall effect of light

The chirality and concentration of biomolecules can be simultaneously determined by weak measurement techniques through spin Hall effect of light (SHEL)effect, which is theoretically and experimentally demonstrated in this paper. The theoretical model concerning the concentration of chiral biomolecules and SHEL has been revealed, taking tryptophan solution as an example, which shows great linearity and the sensitivity can reach to 13 um/mdeg which just use SiO2 as the reflective interface. Moreover, by inducing a film such as Ce1Y2Fe5O12(CeYIG) film with the optimized thickness, the sensitivity is increase to 23 um/mdeg for the refractive index gradient formed by the film enhances SHEL. In order to distinguish these isomers, these two isomers are called L-left-handed and D-right-handed, because the interaction between these chiral biomolecules and polarized light causes the plane of polarization to rotate in the opposite direction. In order to verify our theoretical results, we experimentally measured the SHEL of L- and D-tryptophan solutions with different concentrations. The results indicate that the sensitivity is 20.5 um/mdeg and 20.6 um/mdeg for L- and D-tryptophan solutions, respectively, and increased 1.75 times by introducing the CeYIG film, which is in good agreement with our theoretical results. At the same time, the theoretical model can obtain impurities content between optical isomers in mixed chiral solution. Our study shows great applying potential of weak measurement techniques in exactly and non-contact measurement for chirality and concentration of biomolecules.

Effects of polar charged amino acids on quantum yield of Tyrosine and Tryptophan

Amino acid fluorescence is of particular interest due to its use in determining structural relationships of proteins. The current research is focused on understanding the role of donating amino acid groups on the natural fluorescence of aromatic amino acids, tyrosine and tryptophan. To do this the percentage volume by volume of two polar amino acids, aspartic acid, and glutamic acid within the vicinity of aromatic amino acids, tyrosine and tryptophan were varied. The fluorescence was then measured, and the quantum yields calculated. The results show that the addition of 3.34 mM (25% vol/vol addition) of either glutamic acid or aspartic acid to a 10.28 mM solution of Tryptophan increased the quantum yield by about the same amount. This increase was significantly greater if both concentrations of glutamic acid and aspartic acid (50% v/v) were combined at the same time. However, using a 25% vol/vol addition of glutamic acid and aspartic acid to tyrosine did not have the same effect on the quantum of tyrosine, with aspartic acid showing a quantum yield increase of 0.178 compared to 0.143 shown by glutamic acid. In addition, the combined effect of both amino acids was not greater. The results indicate that tyrosine emits photons in the presence of either glutamic acid or aspartic acid disproportionally while tryptophan does not. On the other hand, the combined effect of both aspartic acid and glutamic acid causes greater photon emission in tryptophan. These results if proven would suggest that photon emission effect of aromatic aminos is greatly influenced by the concentration and types of amino acids within the vicinity of emitting amino acids.

Influence of natural L-amino acids on the interfacial tension of an oil-water system and rock wettability alterations

The cost and environmental effects of most conventional surfactants have increased the search for a greener and cheaper alternative for enhanced oil recovery (EOR) applications. The natural L-amino acids are amphoteric compound with excellent surface properties that have found versatile applications in the pharmaceutical, cosmetic, and food industries. These amino acids exhibited amphipathic nature due to their hydrophobic alkyl chain and charged head. Hence, they could form contacts with both polar and non-polar surface ligands simultaneously. In this study, four amino acids, namely L-arginine, L-lysine, L-methionine, and L-tryptophan, were investigated to access their ability to reduce the interfacial tension (IFT) and alter the wettability of oil-wet sandstone rock. The pendant/rising drop and sessile drop method were employed in IFT and contact angle measurements. The stability of amino acids in brine and the compatibility with other EOR chemicals were tested through visual observation of the precipitates and cloudiness. With the exception of L-tryptophan, the compatibility and stability results revealed that all the amino acids have high salt tolerance above 25 wt% sodium chloride (NaCl). The solutions of amino acids and chemicals such as cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), hydrolyzed polyacrylamide (HPAM), alkali, and alkali-polymer were transparent even at 80 °C.The IFT test results showed that the amino acids significantly reduced the crude oil-water IFT compared to the IFT of n-decane-water system. The IFT between deionized water and crude oil decreased by 76.61% and 55.24%, 24.79%, 45.23% in the presence of L-arginine, lysine, methionine, and tryptophan solutions, respectively. Addition of 0.01 wt% L-arginine, L-lysine and L-methionine to 0.1 wt% SDS reduced the IFT by 28.3%, 15.6%, and 10.97% respectively. The contact angle test showed that L-amino acids have great potential as wettability modifying agents. The contact angles reduced with the decreasing pH (potential of hydrogen) and with increasing brine concentrations from 1 to 4 wt%. Specifically, the contact angle reduced from 92.3°, 91.42°, 89.81° and 92.95 to 47.24°, 53.9°, 58.9° and 59.0° for L-arginine, L-lysine, L-methionine and L-tryptophan, respectively at a pH of about 3. The study suggests that the L-amino acids could be a potential EOR agent in a high saline environment, mainly as a wettability modifier, as well as additives to improve the surface activity of the conventional surfactant and EOR chemicals.

«Электронный язык» на основе модифицированных полиариленфталидами электродов для распознавания модельных растворов триптофана

The effect of auxiliary substances in the composition of the dosage form (sucrose, lactose, talc and starch) on the voltammetric registration of tryptophan using two- and threesensor systems such as "electronic tongue" based on glassy carbon electrodes modified with polyarylenephthalides was studied. For the subsequent chemometric processing of the obtained data, the principal component analysis and soft independent modeling of class analogies were used. It has been shown that electrodes modified with various polyarylenephthalides have cross-sensitivity to four model solutions of tryptophan, each of which contains a certain auxiliary component. When the modified electrodes are used separately, the analyzed model solutions of tryptophan form clusters on the score plots of PCA that intersect with each other, which does not allow them to be reliably recognized. However, the combination of modified electrodes into two- and three-sensor systems due to cross-sensitivity makes it possible to obtain the PCA score plots, in which clusters of samples of model solutions of tryptophan intersect less or do not intersect at all, and, therefore, to recognize them with a high probability. In almost all cases of using two- and three-sensor systems, the classification sensitivity is 100%. When using modified electrodes separately, the specificity of the classification varies from 20 to 100%, when using two-sensor systems – from 50 to 100%. The specificity of the classification when using a three-sensor system is not less than 90%. The proposed sensor system, after additional studies, can be used to recognize medicines containing the same active substance, but different auxiliary components, and will also allow detecting the presence of impurities in medicines.

Impact of Local Electrostatics on the Redox Properties of Tryptophan Radicals in Azurin: Implications for Redox-Active Tryptophans in Proton-Coupled Electron Transfer.

Tyrosine and tryptophan play critical roles in facilitating proton-coupled electron transfer (PCET) processes essential to life. The local protein environment is anticipated to modulate the thermodynamics of amino acid radicals to achieve controlled, unidirectional PCET. Herein, square-wave voltammetry was employed to investigate the electrostatic effects on the redox properties of tryptophan in two variants of the protein azurin. Each variant contains a single redox-active tryptophan, W48 or W108, in a unique and buried protein environment. These tryptophan residues exhibit reversible square-wave voltammograms. A Pourbaix plot, representing the reduction potentials versus pH, is presented for the non-H-bonded W48, which has potentials comparable to those of tryptophan in solution. The reduction potentials of W108 are seen to be increased by more than 100 mV across the same pH range. Molecular dynamics shows that, despite its buried indole ring, the N-H of W108 hydrogen bonds with a water cluster, while W48 is completely excluded from interactions with water or polar groups. These redox properties provide insight into the role of the protein in tuning the reactivity of tryptophan radicals, a requirement for controlled biological PCET.

Identification of important precursors and theoretical toxicity evaluation of byproducts driving cytotoxicity and genotoxicity in chlorination

Chlorination, the most widely used disinfection process for water treatment, is unfortunately always accompanied with the formation of hazardous disinfection byproducts (DBPs). Various organic matter species, like natural organic matter (NOM) and amino acids, can serve as precursors of DBPs during chlorination but it is not clear what types of organic matter have higher potential risks. Although regulation of DBPs such as trihalomethanes has received much attention, further investigation of the DBPs driving toxicity is required. This study aimed to identify the important precursors of chlorination by measuring DBP formation from NOM and amino acids, and to determine the main DBPs driving toxicity using a theoretical toxicity evaluation of contributions to the cytotoxicity index (CTI) and genotoxicity index (GTI). The results showed that NOM mainly formed carbonaceous DBPs (C-DBPs), such as trichloromethane, while amino acids mainly formed nitrogenous DBPs (N-DBPs), such as dichloroacetonitrile (DCAN). Among the DBPs, DCAN had the largest contribution to the toxicity index and might be the main driver of toxicity. Among the precursors, aspartic acid and asparagine gave the highest DCAN concentration (200 g/L) and the highest CTI and GTI. Therefore, aspartic acid and asparagine are important precursors for toxicity and their concentrations should be reduced as much as possible before chlorination to minimize the formation of DBPs. During chlorination of NOM, tryptophan, and asparagine solutions with different chlorine doses and reaction times, changes in the CTI and GTI were consistent with changes in the DCAN concentration.

Electro-Driven in Situ Construction of Functional Layer Using Amphoteric Molecule: The Role of Tryptophan in Ion Sieving

The permselective separation of monovalent ions from the solution with the coexistence of multivalent ions was required in the industry of the wastewater treatment and resource recycling. Here, in this work, a novel electro-driven in situ modification method was utilized in the positively charged tryptophan solution to prepare highly permselective cation exchange membrane (CEM). We have optimized the process conditions and discussed the modification mechanism by drawing upon the fouling phenomenon in the membrane separation processes. A series of conventional characterization methods such as scanning electron microscopy (SEM), attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR), and electrochemical impedance spectroscopy (EIS) were used to investigate the structure and performance changes after the modification. The SEM-energy-dispersive X-ray (SEM-EDX) was introduced for analyzing the transfer of Na+ and Mg2+ in the functional modification layer and the membrane matrix, thus illustrating the sieving mechanism for the modified membranes. The resultant membranes were observed to have the changed interstructure and the multivalent-ion-repulsive modification layer. Due to the channel filling and electrostatic repulsion effect, the electro-driven tryptophan endowed the pristine CMX with superior monovalent cation permselectivity (PMg2+Na+: 6-35), which was higher than that of pristine CMX and the commercial Neosepta CIMS. It confirms that the proposed electro-driven tryptophan in situ modification method could effectively help improve the membrane permselectivity by structurally forming stable crystallization on and within the membrane, which provides a feasible choice for the cation exchange membrane modification.

Investigation of tryptophan-related yellowing in parenteral amino acid solution: Development of a stability-indicating method and assessment of degradation products in pharmaceutical formulations

Parenteral amino acid solutions containing tryptophan tend to develop a yellow colouration upon storage. Hence, the aim of the present study was to find out whether tryptophan degradation products are the reason for the yellowing. The degree of discolouration and tryptophan degradation was examined by visual examination and UV/Vis measurements with respect to oxygen presence, pH value, and duration of steam sterilization. LC-UV analyses of autoclaved tryptophan solutions indicated eight degradation products, namely R,R/R,S 2-amino-3-(oxoindolin-3-yl)propanoic acid, R,R/R,S 2-amino-3-hydroxy-2-oxoindolin-3-yl)propanoic acids, cis/trans 3a-hydroxy-1,2,3,3a,8,8a-hexahydropyrrolo[2,3-b]indole-2-carboxylic acid, N´-formylkynurenine, and kynurenine. The proposed degradation products were confirmed by spiking of synthesized degradation products and LC-UV/MS analyses. The LC-UV analysis method was optimized and validated according to the ICH guideline Q2 (R1). Tryptophan stability in commercially available parenteral amino acid formulations was evaluated over a storing period of 12 months in two common types of primary packaging after autoclave procedure.

Raman and Quantum Yield Studies of Trp48-d5 in Azurin: Closed-Shell and Neutral Radical Species.

Isotopologues are valuable vibrational probes that shift features in a vibrational spectrum while preserving the electronic structure of the molecule. We report the vibrational and electronic spectra of perdeuterated tryptophan in solution (l-Trp-d5), as Trp48-d5 in azurin, and as the photogenerated neutral tryptophan radical, Trp48-d5•, in azurin. The UV resonance Raman bands of the perdeuterated closed-shell tryptophan in solution and in azurin are lower in frequency relative to the protiated counterpart. The observed decrease in frequencies of l-Trp-d5 bands relative to l-Trp-h5 enables the analysis of vibrational markers of other amino acids, e.g., phenylalanine, that overlap with some modes of l-Trp-h5. The Raman intensities vary between l-Trp-d5 and l-Trp-h5; these differences likely reflect modifications in normal mode composition upon perdeuteration. Analysis of the W3, W6, and W17 modes suggests that the W3 mode retains its utility as a conformational marker; however, the H-bond markers W6 and W17 appear to be less sensitive upon perdeuteration. The neutral tryptophan radical, Trp48-d5•, was generated in azurin with a slightly lower radical quantum yield than for Trp48-h5•. The visible resonance Raman spectrum of Trp48-d5• is different from that of Trp48-h5•, especially in terms of relative intensities, and all assignable peaks decreased in frequency upon perdeuteration. The absorption and emission spectra of the perdeuterated closed-shell and radical species exhibited hypsochromic shifts of less than 1 nm relative to the protiated species. The data presented here indicate that l-Trp-d5 is a valuable probe of vibrational structure, with minimal modification of photoreactivity and photophysics compared to l-Trp-h5.