Low field CIDNP of amino acids and proteins: characterization of transient radicals and NMR sensitivity enhancement

A new method for measuring and exploiting the magnetic field dependence of chemically induced dynamic nuclear polarization (CIDNP) is described. A solution of an amino acid or protein together with a flavin photosensitizer is irradiated with laser light at a position in the bore of a superconducting NMR magnet where the field is between 0.1 T and 7.0 T. The polarized sample is then transferred by rapid injection into an NMR tube at the centre of the magnet (at 9.4 T), where the spectrum is recorded. The observed ¹H CIDNP field dependence of tyrosine agrees well with the diffusion model of the radical pair mechanism. The field dependence of histidine, tryptophan and methionine CIDNP allows the g values of the transient radicals responsible for the polarization to be determined. Experiments in which amino acids compete for the photoexcited flavin indicate that methionine residues could be used as probes of surface accessibility, especially if the polarization is generated in low fields (～ 0.7 T) and detected in high fields (≥ 9.4 T). Possible extensions of the technique to study protein folding and the structures of partially denatured states of proteins are discussed.     

Reduction of Guanosyl Radicals in Reactions with Proteins Studied by TR-CIDNP

Pulsed-field-gradient nuclear magnetic resonance (NMR) combined with time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP) was applied to study the reduction of guanosyl radicals in reactions with the proteins hen egg white lysozyme (HEWL) and bovine α-lactalbumin (BLA) in their native state. Guanosyl radicals were generated photochemically in the reaction of guanosine-5′-monophosphate with photosensitizer, triplet-excited 2,2′-dipyridyl. In this reaction, at pH 5 guanosyl cation radical is formed, which deprotonates to yield the neutral guanosyl radical. To minimize the contribution of the cation radical, phosphate buffer was added, which accelerates the deprotonation of guanosyl cation radical. From model simulations of CIDNP kinetics the rate constants of the reduction were found to be (3.1 ± 0.5) × 10⁷ M^?1s^?1 for HEWL and (1.6 ± 0.4) × 10⁷ M^?1s^?1 for BLA. Also, experiments were carried out at the conditions for denatured HEWL, i.e., at 50 °C in the presence of 10 M urea-d₄. The rate constant of the reduction of guanosyl radical in this case was (3.6 ± 0.5) × 10⁸ M^?1s^?1.     

Photooxidation of Histidine by 3,3′,4,4′-Benzophenone Tetracarboxylic Acid in Aqueous Solution: Time-Resolved and Field-Dependent CIDNP Study

The photooxidation reaction between 3,3′,4,4′-benzophenone tetracarboxylic acid (TCBP) and l-histidine (His) has been investigated in neutral aqueous solution using the technique of chemically induced dynamic nuclear polarization (CIDNP). Relative values of ¹³C isotropic hyperfine couplings in the TCBP and His radicals were obtained from the ¹³C-time-resolved CIDNP spectrum, recorded during the photoreaction of TCBP with His at natural abundance of the magnetic isotope ¹³C. Good agreement was found for the hyperfine coupling constants of the TCBP ketyl radical calculated using methods of density functional theory, and those obtained from the ¹³C-time-resolved CIDNP spectrum. The mechanism of the quenching reaction of triplet-excited TCBP by His in neutral aqueous solution was established. ¹H CIDNP field dependencies for the photoreaction of TCBP with His were obtained and the g-factor of the histidyl radical was found.     

Conformational Transitions in <Emphasis Type="Italic">Ariesaema curvatum</Emphasis> Lectin: Characterization of an Acid Induced Active Molten Globule

Biophysical characterization of a lectin from Ariesaema curvatum (ACL) was carried out using steady state as well as time resolved fluorescence and CD spectroscopy under various denaturing conditions. An intermediate with altered tryptophan microenvironment was detected in the phase diagram, which exibited pronounced secondary structure and hemagglutinating activity in presence of 0.25 M Gdn–HCl. An acid induced molten- globule like structure possessing activity and higher thermostability was detected. Transition to the molten globule state was reversible in nature. The lectin retained hemagglutinating activity even after incubation at 95 °C. Both chemical and thermal unfolding of the lectin were found to consist of multistate processes. Fluorescence quenching of ACL was strong with acrylamide and KI. The single tryptophan was found to be surrounded by high density of the positively charged amino acid residues as shown by a ten fold higher K_sv for KI compared to that for CsCl. The average lifetime of tryptophan fluorescence increased from 1.24 ns in the native state to 1.72 ns in the denatured state.     

Quantitative Approach to CIDNP in Proteins with Several Polarizable Residues on the Surface

A quantitative theoretical approach to protein chemically induced dynamic nuclear polarization (CIDNP) is formulated in the present work, which is based on the theory of sterically specific chemical reactions in liquids. Kinetic equations for polarization of all CIDNP-active accessible amino acid residues are presented. Relations between the kinetic parameters and the total side-chain accessibility (TSA) values are established. Numerical calculations of the CIDNP kinetics are also shown to demonstrate how the time behavior of polarization depends on TSA. The present theoretical approach was applied to model time-resolved protein CIDNP data obtained for Tyr59 and His68 of ubiquitin in the native state. Comparison of theoretical predictions with the experimental data confirms the accuracy of the approach.     

激光引发自由基反应磁效应的光谱学研究

“动态自旋化学”(dynamic spin chemistry)作为一门新兴的交叉研究领域,其重要性已得到广泛的共识。涉及的研究内容包括： 化学反应的磁效应(MFE)、同位素效应(MIE)、化学诱导动态核极化(CIDNP)和化学诱导动态电子极化(CIDEP)。文章简要介绍了激光引发自由基反应的磁效应发展历史及其光谱学研究方法。分析并总结了自由基反应磁效应产生的原因、单-三转换理论及磁效应机理。同时,也为国内同行介绍了自由基反应磁效应研究新的发展动态。     

Deprotonation of Transient Guanosyl Cation Radical Catalyzed by Buffer in Aqueous Solution: TR-CIDNP Study

The reaction of deprotonation of the guanosyl cation radical formed in the photoinduced reaction of guanosine monophospate (GMP) with triplet 2,2??-dipyridyl-d₈ is studied in aqueous solution by time-resolved chemically induced dynamic nuclear polarization (TR-CIDNP). In the course of the cyclic photoreaction, spin-polarized products are generated. Their polarization patterns that reflect the properties at the radical stage are analyzed using high-resolution nuclear magnetic resonance. The identification of transient radicals contributing to the polarization kinetics is based on its sensitivity to the degenerate electron exchange reaction of transient radicals with the parent diamagnetic molecules. Degenerate electron exchange is allowed only for the cation radical and manifests itself in the fast decay of the CIDNP signal in time with the rate of decay proportional to the concentration of parent GMP molecules. Because the formation of the neutral transient radical stops the exchange, the deprotonation changes the CIDNP kinetics from a decaying to a growing one. The rate constant of deprotonation, k _d, was obtained from modeling of CIDNP kinetics data with taking into consideration the difference of the CIDNP enhancement factors for neutral and cation guanosyl radicals. The value obtained at pH^* 5 for k _d?=?1?×?10⁶?s^?1 is consistent with the proton dissociation constant of the radical (pK _a?=?3.9). The linear dependence of the deprotonation rate on the buffer concentration is revealed for phosphate, formate, and acetate. Deprotonation is catalyzed by the buffer to a degree that depends on the difference in pK _a value of the buffer and the guanosyl cation radical in full accordance with Eigen??s model.     

A study of the primary photochemical processes occurring in proteins

Solutions of aromatic amino acids irradiated with ultraviolet light at 77° K have been found to form products responsible for the photofading of the fluorescence of the initial molecules. Luminescent in the yellow-green portion of the spectrum, these products are considered to be free radicals. In the case of tyrosine, the luminescence excitation spectrum appears to be determined by the phenoxy radical. The formation of tryptophan radicals involves an intermediate stage in which the initial molecule probably passes through a triplet state.     

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.     

Influence of nitroxides on CIDNP of micellised radical pairs and short-lived biradicals

The effects of stable nitroxide radicals on stimulated nuclear polarization (SNP) and chemically induced dynamic nuclear polarization (CIDNP) in short-lived consecutive biradicals and radical pairs in homogeneous solutions as well as spin-correlated radical pairs in micelles were studied in high and low magnetic fields. It is shown that experimentally observed effects of nitroxide additions on CIDNP and SNP can be well simulated taking into account only the increase in the rates of relaxation in the paramagnetic species constituting radical pairs or biradicals. Effects of coherent spin evolution in three-spin systems under study seem to be of negligible importance.     

Charge transfer reactions from tryptophan and tyrosine to sulfur‐centered dimer radical cation in aqueous –sulfuric acid medium: a pulse radiolysis study

Kinetics and mechanism of one‐electron oxidation of N‐acetyl methionine (NAM), tryptophan (TrpH), tyrosine (TyrOH), and phenylalanine (Phe) have been studied in 33% v/v H₂SO₄ solution. The solvent radical (SO₄^?¯) oxidized NAM, TrpH, TyrOH, and Phe to produce NAM₂^?+ (480 nm), TrpH^?+ (330, 580 nm), TyrO^? (350, 410 nm), and Phe(?H)^? (320 nm), with rate constants (10⁹ M^?1 s^?1) 0.6, 2.7, 3.9, 1.6, respectively. Time resolved radical transformation from NAM₂^?+ to TrpH^?+ and TyrO^? have been observed to occur with k(10⁸ M^?1 s^?1) = 3.60 and 0.35, respectively. However, NAM₂^?+ to Phe(?H)^? and TrpH^?+ to TyrO^? radical transformations have not been observed in this medium. The study shows the kinetics and mechanism of oxidation of some amino acids in strong acidic solutions. To the best of our knowledge, radical cations of amino acids and electron transfer reactions between them could be studied in strong acidic solutions for the first time. Copyright © 2016 John Wiley & Sons, Ltd.     

导数荧光-偏最小二乘法同时测定注射液中色氨酸、酪氨酸和苯丙氨酸

本文用偏最小二乘 (PLS)法对苯丙氨酸、酪氨酸、色氨酸混合体系的导数荧光光谱进行解析 ,提出了同时测定三种氨基酸的计算分析方法。以pH 7 4的磷酸缓冲溶液为介质 ,以 2 16 6nm为激发波长 ,对复合氨基酸注射液进行三组分同时测定 ,相对误差均在± 7 2 %以内。     

Coherent Polarization Transfer Effects Are Crucial for Interpreting Low-Field CIDNP Data

In this work we demonstrate that low-field chemically induced dynamic nuclear polarization (CIDNP) is strongly affected by re-distribution of polarization, which is formed in the course of spin evolution in transient radical pairs, in diamagnetic reaction products. This phenomenon is of importance when the spins of the reaction product are coupled strongly meaning that spin–spin interactions between them are comparable to the differences in their Zeeman interactions with the external magnetic field. In this case, polarization transfer relies on a coherent mechanism; as a consequence, spins can acquire significant polarization even when they have no hyperfine coupling to the electron spins in the radical pairs, i.e., cannot be polarized directly by CIDNP. This is demonstrated by taking CIDNP of n-butylamine as an example: in this case only the α-CH₂ protons are polarized directly, which is confirmed by high-field CIDNP, whereas the β-CH₂, γ-CH₂ and δ-CH₃ protons get polarized only indirectly due to the transfer of polarization from the α-CH₂ protons. These results show that low-field CIDNP data should be interpreted with care to discriminate between the effects of spin evolution in transient radical pairs and in diamagnetic reaction products.     

Steady State and Time Resolved Fluorescence Quenching and Chemical Modification Studies of a Lectin from Endophytic Fungus <Emphasis Type="Italic">Fusarium solani</Emphasis>

The solute quenching studies of a lectin from endophytic fungus Fusarium solani were carried out using different quenchers such as acrylamide, succinimide, potassium iodide and cesium chloride. The lectin showed emission maximum at 348 nm indicating relative exposure of tryptophan. The quenchable fraction of the fluorophore was 100% with acrylamide, whereas it was only 50% with succinimide. The ionic quenchers iodide and cesium showed opposite effects at different pH. In the case of cesium, raising the pH resulted in increased quenching and accessibility of typtophan residue, while the iodide showed just opposite effect. These studies showed that the single tryptophan residue of the lectin (per monomer) is relatively exposed, and might be in the vicinity of positively charged amino acid residues. Various amino acids of the F. solani lectin were modified using different reagents to obtain information about the hemagglutinating site. The chemical modification studies suggested tyrosine residues can be modified using N-acetylimidazole, which results in complete loss of hemagglutination activity of the lectin. Kinetics of chemical modification suggested involvement of only 2 tyrosine residues. Modification of arginine, cysteine, histidine, lysine, aspartate, glutamate and tryptophan did not result in loss of hemagglutinating activity of the lectin.     

The CIDNP effect in plastic crystals

The CIDNP effects in the photolysis reactions of some aldehydes and ketones (including linear, aromatic and cyclic) in plastic crystals of cyclohexane have been discovered and studied. In going from liquid to solid solutions, the change of polarization sign is observed for some substances investigated. The CIDNP effect for a simple model for a relative motion of atoms of radicals possessing spin density (translational diffusion in a restricted volume) has been calculated. The results observed are interpreted in the framework of the radical pair mechanism of CIDNP effect formation.     

乙腈-水和乙二醇-水均相溶液中光诱导杜醌激发三重态与色氨酸、酪氨酸的氢原子转移反应

本文用激光闪光光解技术研究了光诱导生物醌杜醌激发三重态(³DQ^*)和色氨酸(Trp)与酪氨酸(Tyr)在乙腈-水(MeCN-H₂O)及乙二醇-水(EG-H₂O)均相溶液中的光化学反应,分析了反应的机理,并基于Stern-Volmer方法测量了反应速率常数. 光解DQ体系可以生成³DQ^*,³DQ^*与Trp、Tyr发生的氢原子转移反应占主导地位. 对于DQ/Trp/MeCN-H₂O和DQ/Trp/EG-H₂O溶液,³DQ^*与Trp反应生成杜醌中性自由基DQH^·、以碳为中心的色氨酸中性自由基Trp^·/NH和以氮为中心的色氨酸中性自由基Trp/N^·. 对于DQ/Tyr/MeCN-H₂O和DQ/Tyr/EG-H₂O溶液,³DQ^*与Tyr反应生成DQH^·和酪氨酸中性自由基Tyr/O^·. ³DQ^*与Trp、Tyr的氢原子转移反应速率常数都在10⁹ L·mol^-1·s^-1量级,反应近似受扩散控制. MeCN/H₂O均相溶液中³DQ^*与Trp、Tyr的反应速率常数要明显高于EG/H₂O均相溶液中的反应速率常数,这与Stokes-Einstein方程定性一致.     

Azobenzene Pd(II) complexes with N^N- and N^O-type ligands

Methods of synthesis of cyclometalated azobenzene palladium(II) complexes of [Pd(N^N)Azb]ClO₄ and [Pd(N^O)Azb]ClO₄ types (where Azb^– is the deprotonated form of azobenzene; N^N is 2NH₃, ethylenediamine, or 2,2′-bipyridine; and (N^O)^– is the deprotonated form of amino acid (glycine, α-alanine, β-alanine, tyrosine, or tryptophan)) are developed. The electronic absorption and the electrochemical properties of these complexes are studied.     

Tryptophan Environment and Functional Characterization of a Kinetically Stable Serine Protease Containing a Polyproline II Fold

The single tryptophan residue from Nocardiopsis sp. serine protease (NprotI) was studied for its microenvironment using steady state and time-resolved fluorescence. The emission maximum was observed at 353 nm with excitation at 295 nm indicating tryptophan to be solvent exposed. Upon denaturation with 6 M guanidinum thiocyanate (GuSCN) the emission maxima was shifted to 360 nm. Solute quenching studies were performed with neutral (acrylamide) and ionic (I^- and Cs⁺) quenchers to probe the exposure and accessibility of tryptophan residue of the protein. Maximum quenching was observed with acrylamide. In the native state, quenching was not observed with Cs⁺ indicating presence of only positively charged environment surrounding tryptophan. However; in denatured protein, quenching was observed with Cs⁺, indicating charge reorientation after denaturation. No quenching was observed with Cs⁺ even at pH 1.0 or 10.0; while at acidic pH, a higher rate of quenching was observed with KI. This indicated presence of more positive charge surrounding tryptophan at acidic pH. In time resolved fluorescence measurements, the fluorescence decay curves could be best fitted to monoexponential pattern with lifetimes of 5.13 ns for NprotI indicating one conformer of the trp. Chemical modification studies with phenyl glyoxal suggested presence of Arg near the active site of the enzyme. No inhibition was seen with soyabean trypsin and limabean inhibitors, while, CanPI uncompetitively inhibited NprotI. Various salts from Hofmeister series were shown to decrease the activity and PPII content of NprotI.     

CIDNP及其在光化学中的应用

化学诱导动态核极化(Chemically Induced Dynamic Nuclear Polarization,简称CIDNP)是一种在化学反应体系中观察到的NMR谱线强度反常的现象(常称之为极化谱,包括增强吸收和发射等)。1967年由Bargon和Fischer^[1] (西德)以及Ward和Lawler^[2] (美国)首先发现的。由于他们所研究的反应体系中涉及自由基,因此最初认为这种谱线强度反常现象是由于自由基中间体中的电子-核交叉弛豫引起的。这种机理通常称为:Overhauser型的动态核极化(Dynamic Nnclear Polarization简称DNP)。但很快就发现它不足以解释实验得到的结果,而CIDNP的命名却是这样因袭下来了。     

Identification of tyrosine in the presence of tryptophan using Cd-enriched colloidal CdS nanoparticles: A fluorescence spectroscopic study

A simpler identification method of tyrosine in the presence of tryptophan using CdS nanoparticles by conventional spectroscopic technique is proposed. Effect of both sulfide-enriched CdS as well as Cd²⁺-enriched CdS on tryptophan is investigated through absorption and emission spectroscopy. Quenching of tryptophan emission obeyed Stern-Volmer relation and was found to be independent of temperature, indicating a possible static quenching. The time-resolved fluorescence decay of tryptophan was minimally affected by sulfide-enriched CdS as well as Cd²⁺-enriched CdS nanoparticles, suggesting quenching to be static. In the presence of Cd²⁺-enriched CdS nanoparticles, the emission of tryptophan in phosphate buffer shows a typical spectral broadening along with a long wavelength increase in fluorescence emission. Additionally, spectra followed a typical isoemissive point at 440 nm when tryptophan alone was there. Similarly, isoemissive point at 340 nm was observed in the case of tyrosine. However, a further red shift of isoemissive point (470 nm) in the mixture of both tyrosine and tryptophan was observed. This observation might make Cd²⁺-enriched CdS nanoparticles useful for using as marker for tyrosine in the presence of tyrptophan.