Photochemical Reactions of Protein Tryptophanyls in the Eye Lenses of Rats in the Presence of Chloroform

The modifying action of chloroform on photochemical reactions induced in the eye lenses of rats by UVB radiation is revealed. In anesthetic concentrations, halogenmethane increases the quantum yield of photodisintegration of protein tryptophanyls with formation of a new photochemical product that absorbs radiation in the UVA range and possesses the photosensitizing activity.     

Intrinsic Fluorescence of Actin

In this work actin is used to illustrate connection of protein fluorescence characteristics with its structure. On one hand, it has been demonstrated what kind of information about the contribution of each tryptophan residues to the bulk fluorescence spectrum can be obtained from the special analysis of protein three-dimensional structure. On the other hand, potentials of intrinsic fluorescence for elucidation of proteins structure, dynamics and processes of folding-unfolding are shown. In particular, using this method a new essentially unfolded kinetic intermediate state of actin was detected and characterized, and the place of inactivated actin and its kinetic predecessor in the process of folding-unfolding was determined. It has been revealed that inactivated actin is not intermediate state between the native and completely unfolded states, as it has been accepted before, but a result of protein misfolding. On the basis of the obtained data a new model of actin folding-unfolding pathway has been proposed.     

Time-resolved fluorescence study of the single tryptophan in thiocyanate and azide derivatives of horseradish peroxidase: Implication for a p H-induced conformational change in the heme cavity

Detailed pH-dependent steady state and picosecond time-resolved tryptophan fluorescence studies on thiocyanate and azide complexes of horseradish peroxidase have been carried out. The fluorescence decay of the single tryptophan in these species was fitted to a discrete three exponential model. Maximum entropy method analysis also gave three distinct regions of lifetime distributions. The fast subnanosecond lifetime component was found to have > 97% amplitude contribution while other two longer lifetime components have small contributions. Small contributions from the nanosecond lifetime components possibly arise from apoprotein impurity or some small amount of disordered heme conformer of the protein. pH dependence of the fast picosecond lifetime components was found to show a systematic behavior which has been interpreted in the light of obligatory conformation change associated with activation of the enzyme at low pH.     

Fermentation characteristics and the dynamic trend of chemical components during fermentation of Massa Medicata Fermentata

As a representative of traditionally fermented Chinese medicine, Massa Medicata Fermentata (MMF) shows the functions of invigorating the spleen and stomach and promoting digestion, which plays an important role in the treatment of gastrointestinal diseases. The fermentation mechanism and the key factors that affect the quality of MMF have not been revealed yet, which has become an urgent issue that limits its clinical application. This article aims to systematically and comprehensively reveal the transformation of physical properties and the dynamic trend of chemical components including substrate components, volatile components, and lactic acid as anaerobic fermentation product during MMF fermentation. Along with obvious hyphae growth observed for MMF, the weight of MMF decreased, and the moisture and temperature increased. Through the quantified 14 components from substrate, ferulic acid increased from 45.53 ± 6.94 to 141.89 ± 78.40 μg/g, while glycosides and phenolic acids declined except caffeic acid. Also, within the 66 volatile components analyzed, alcohols and acids increased, while aldehydes and ketones decreased. Lactic acid was not detected in the fermentation substrate, but an apparent increase in lactic acid content was observed along with the increased fermentation days, resulting in 2.54 ± 0.15 mg/g on day 8. Based on the tested components, the fermentation process of MMF was discriminated into three distinct stages by principal component analysis, and an optimal fermentation time of four days was proposed. The results of this study will be of great significance to clarify the characteristics of fermentation and conduce to improving quality standards of MMF.     

Distance-dependent fluorescence quenching ofN-acetyl-l-tryptophanamide by acrylamide

We examined the time-dependent intensity decays ofN-acetyl-l-tryptophanamide (NATA) when collisionally quenched by acrylamide in propylene glycol over a range of temperatures. The intensity decays of NATA became increasingly heterogeneous in the presence of acrylamide. The NATA intensity decays were not consistent with the Collins-Kimball radiation boundary condition (RBC) model for quenching. The steady-state Stern-Volmer plots show significant upward curvature, and quenching of NATA by acrylamide was observed even in vitrified propylene glycol, where translational diffusion cannot occur during the lifetime of the excited state. These frequencydomain and steady-state data indicate a through-space quenching interaction between NATA and acrylamide, and the results are consistent with a rate constant for quenching that depends exponentially on the fluorophore-quencher separation distance. The exponential distance-dependent rate of quenching also explains the upward curvature of the Stern-Volmer plot, and the steady-state data aid in determining the interaction distance between NATA and acrylamide. These results suggest that the distance-dependent quenching rates need to be considered in the interpretation of acrylamide quenching of proteins.     

Electrocatalysis of Tryptophan at Multi-Walled Carbon Nanotube Modified Electrode

Acid-treated multi-walled carbon nanotubes (MWNTs) were immobilized on the surface of a glassy carbon electrode to form an MWNT-modified electrode. The electrocatalytic response of the modified electrode towards tryptophan (Trp) was investigated by cyclic voltammetry (CV) and differential pulse voltammetry (DPV). The results demonstrated that the modified electrode exhibited a high degree of catalytic activity towards the oxidation of Trp. An oxidation peak was obtained in Trp solution at the MWNT-modified electrode. Compared with a bare electrode, the peak current had obviously increased, and the peak potential had shifted in a negative direction. However, under the same conditions, no response was observed for other amino acids. The oxidation peak currents were proportional linearly to the concentration of Trp, a property which could be utilized to detect Trp. The determination conditions, such as the concentration, the composition and the pH values of the supporting electrolyte, accumulation time, as well as scan rate etc. were optimized. Under the chosen conditions, the DPV peak current is linear to the concentration of Trp in the range of 2.5×10^–7 to 1.0×10^–4molL^–1, and the detection limit is 2.7×10^–8molL^–1. Moreover, the detection is free of interference from other amino acids. The modified electrode has been successfully applied to determine the concentration of Trp in composite amino acid injections, and it displays excellent repeatability and higher sensitivity.     

阴阳离子型表面活性剂双水相萃取色氨酸衍生物和牛血清白蛋白

溴化十二烷基三乙胺（Ｃ１２ＮＥ）和十二烷基硫酸钠（ＳＤＳ）在一定条件下混合可以形成具有清晰界面的两个水相，称为阴阳离子型表面活性剂双水相。作者研究了利用该双水相萃取３种色氨酸衍生物和牛血清白蛋白的可能性．文中采用工作曲线校正扣除表面活性剂背景的影响，萃取结果准确可信。     

Simultaneous determination of tryptophan,kynurenine, kynurenic acid,xanthurenic acid and 5‐hydroxytryptamine in human plasma by LC‐MS/MS and its application to acute myocardial infarction monitoring

Reliable methods for the determination of tryptophan and its metabolites are vital to the monitoring of biochemical states during the initiation and progression of cardiovascular disease. In the present study, a single‐run liquid chromatography–tandem mass spectrometry (LC‐MS/MS) method was developed for the simultaneous determination of tryptophan (Trp) and its metabolites, including kynurenine (Kyn), kynurenic acid (KA), xanthurenic acid (XA) and 5‐hydroxytryptamine (5‐HT), in human plasma. The plasma samples were prepared using a protein precipitation approach, and the chromatographic separation was performed by gradient elution on a C₁₈ column within a total analysis time of 3.5 min. The calibration ranges were 40–20,000 ng/mL for Trp, 4–2000 ng/mL for Kyn, 0.2–100 ng/mL for KA, 0.4–200 ng/mL for XA and 1–500 ng/mL for 5‐HT, and the precision and accuracy were acceptable. The evaluation of recovery and internal standard‐normalized matrix effect proved that the sample preparation approach was effective and the matrix effect could be negligible. The newly developed method was successfully applied to the analysis of plasma samples from healthy individuals and myocardial infarction patients. The findings suggested that the plasma concentrations of Trp, Kyn, 5‐HT as well as the concentration ratios of Kyn/Trp and Trp/5‐HT might serve as biomarkers for the monitoring of acute myocardial infarction.     

Development of simultaneous analysis of tryptophan metabolites in serum and gastric juice – an investigation towards establishing a biomarker test for gastric cancer diagnosis

To evaluate changes in tryptophan metabolism and discover diagnostic biomarkers for gastric cancer, a quantitative method was developed for tryptophan and its seven metabolites (indole‐3‐lactic acid, anthranilic acid, serotonin, nicotinic acid, kynurenic acid, kynurenine and 3‐indoxyl sulfate) in both human serum and gastric juice using liquid chromatography–tandem mass spectrometry (LC–MS/MS). Serum and gastric juice were prepared with a simple protein precipitation using aqueous 0.1% formic acid and acetonitrile. As a result, it was found that the kynurenine pathway of tryptophan metabolism was activated in gastric cancer and that the metabolic ratio of kynurenine/tryptophan, which reflects the enzyme activity of indoleamine‐2,3‐dioxygenase, was associated with the observed metabolic changes. Finally, the investigation of tryptophan metabolites, especially kynurenic acid, in serum and gastric juice might serve as biomarkers for gastric cancer. The findings in this study provide critical information of tryptophan metabolism which can be applied to a serum‐based diagnostic test for gastric cancer.     

Ultrafast Fluorescence Spectroscopy via Upconversion and Its Applications in Biophysics

In this review, the experimental set-up and functional characteristics of single-wavelength and broad-band femtosecond upconversion spectrophotofluorometers developed in our laboratory are described. We discuss applications of this technique to biophysical problems, such as ultrafast fluorescence quenching and solvation dynamics of tryptophan, peptides, proteins, reduced nicotinamide adenine dinucleotide (NADH), and nucleic acids. In the tryptophan dynamics field, especially for proteins, two types of solvation dynamics on different time scales have been well explored: ~1 ps for bulk water, and tens of picoseconds for “biological water”, a term that combines effects of water and macromolecule dynamics. In addition, some proteins also show quasi-static self-quenching (QSSQ) phenomena. Interestingly, in our more recent work, we also find that similar mixtures of quenching and solvation dynamics occur for the metabolic cofactor NADH. In this review, we add a brief overview of the emerging development of fluorescent RNA aptamers and their potential application to live cell imaging, while noting how ultrafast measurement may speed their optimization.