Study On The Enzyme-like Activity Of Acidic Amino Acids And Nucleotide And Their Analytical Applications

Horseradish peroxidase?HRP?as a natural enzyme has been extensively used in biosensing,bioremediation,biocatalysis and clinical diagnosis due to its high catalytic efficiency and substrate specificity.Nevertheless,the practical application of HRP suffers from high cost,expensive preparation and purification as well as easy to denaturation and deactivation under harsh reaction conditions on account of its protein structure and functions.Since the pioneering discovery of Fe₃O₄ magnetic nanoparticles with peroxidase-like activity in 2007 by Yan's group,most nanomaterials have evoked increased interest to mimick peroxidase with the development of nanotechnology,but little attention has been paid to small molecular mimic enzymes.Recently,some small molecules,such as two ethylamine four acetic acid,fluorescein and its derivatives,have been found to possess peroxidase-like activity.Herein,we found that natural molecular acidic amino acids?L-glutamic acid?L-Glu?and L-aspartic acid?L-Asp??,adenosine triphosphate?ATP?,guanosine triphosphate?GTP?and its nucleotide analogues have the catalytic activity similar to the natural enzyme,and the catalytic mechanism has been studied in detail.At the same time,we constructed a series of biosensors for sensitive and selective colorimetric assay of glucose,alkaline phosphatase and alpha fetoprotein.Besides,ATP as antioxidant enzyme can protect cells against endogenous H₂O₂-induced oxidative damage.The research work is mainly divided into the following sections:1.The peroxidase-like activity of acidic amino acid and its analytical applicationHerein we found that acidic amino acids including L-glutamic acid?L-Glu?and L-aspartic acid?L-Asp?exhibited an intrinsic peroxidase-like activity,endowing acidic amino acids with the capability of catalyzing the oxidation of the peroxidase substrates 3,3?,5,5?-tetramethylbenzidine?TMB?to produce color reaction in the presence of H₂O₂ under acid condition.The elements analysis of L-Glu and L-Asp were conducted by inductively coupled plasma-mass spectrometry?ICP-MS?,and the metal elements with content more than 0.001%?w/w?were determined under the experiment conditioin.The results showed that the catalytic activity of the acidic amino acid was derived from itself rather than trace metal element.We further studied the effect of side-chain dependent composition on catalytic activity of acidic amino acid,and the results showed that only the side chain with carboxyl amino acids can mimick peroxidase.Reactive oxygen species inhibition experiments and electron spin resonance spectroscopy?ESR?experiments indicated that the reactive oxygen intermediates produced by acidic amino acid mediated catalytic reactions is hydroxyl radical.We further studied the catalytic process by enzyme kinetics and quantum theory.The results showed that the catalytic process follow the Michaelis-Menten model,and the carboxyl group may be the binding site of the substrate H₂O₂,which was consistent with the previous literature.Based on the peroxidase-like activity of acidic amino acids,a sensing platform was constructed for colorimetric detection of glucose,which has high sensitivity and selectivity,and the proposed method had been successfully used for the detection of glucose in human blood samples.Meanwhile,it can also be used to evaluate the antioxidant behaviour.2.The peroxidase-like activity of ATP and its application in detection of glucoseAdenosine triphosphate?ATP?,an important biomolecule,is the most direct source of energy in organisms.It plays an important role in many cellular metabolic and biochemical pathways,such as DNA replication,biomolecular synthesis,muscle contraction and signal transduction.Herein,we showed that ATP possessed peroxidase-like activity,catalyzing H₂O₂ to oxidize TMB to produce a color reaction under acidic conditions.At the same time,high performance liquid chromatography?HPLC?pointed to that ATP changed little in either quality or quantity during the reaction and supported that ATP indeed act as a catalyst rather than a reactant to participate in the TMB-H₂O₂ reaction.In addition,we have demonstrated that the reactive oxygen species produced in the TMB-H₂O₂ reaction catalyzed by ATP is hydroxyl radical,and the catalytic process followed the Michaelis-Menten model.In the preparatory phase of glycolysis,glucose is phosphorylated by a family of enzymes called hexokinases?HK?to form glucose 6-phosphate with Mg²⁺ion as an auxiliary factor,and in this process,ATP is consumed and converted to adenosine diphosphate?ADP?.At the same condition,ADP show barely ability of facilitating the chromogenic reaction between TMB and H₂O₂.Based on this principle,a biosensor with high sensitivity and selectivity for glucose detection was constructed.The developed colorimetric sensing system was also apllied for glucose detection in human serum.3.The catalase-like activity of ATP and its protective effects on mitochondria and cellsHerein,ATP was found to show the catalase mimetic activity under physiological conditions,which can catalyze the decomposition of H₂O₂ to produce water and oxygen.ATP is mainly synthesized in mitochondria,which is the main site of energy production in cells and an important organelle associated with aerobic respiration,energy conversion and cell apoptosis.Therefore,mitochondria are closely related to the normal function of cells,while the stability of mitochondrial membrane potential is beneficial to maintain the normal physiological function of mitochondria.Excessive accumulation of H₂O₂ in mitochondria leads to a decrease in mitochondrial membrane potential and resulting to mitochondria damage.In this perspective,we envisaged that ATP can act as catalase for the opportune control of mitochondria H₂O₂ levels under pathophysiological conditions and the results demonstrated that oxidative damage to mitochondria caused by high concentration of H₂O₂ can be robustly eliminated.We further demonstrated that ATP as catalase can protect cells against endogenous H₂O₂-induced oxidative damage.4.The peroxidase-like activity of GTP and its application in alkaline phosphatase detection and immunoassayGuanosine triphosphate?GTP?,a substrate for RNA synthesis,plays a pivotal role in life-form metabolism at the cellular level.It plays an important role in regulating cell processes and a series of biochemical pathways.Herein,we showed that GTP exhibited an intrinsic peroxidase-mimic activity,accelerating H₂O₂-mediated oxidation of 3,3?,5,5?-tetramethylbenzidine?TMB?to produce a color reaction.Ultra-performance liquid chromatography-mass spectrum?UPLC-MS?pointed to that GTP changed little in either quality or quantity during the reaction and supported that GTP indeed act as a catalyst to mimic peroxidase.In addition,electron spin resonance?ESR?experiments demonstrated that GTP exhibited catalytic ability to decompose H₂O₂ into·OH radicals,and the enzyme kinetics study showed that the catalytic process is follow the Michaelis-Menten model.Alkaline phosphatase?ALP?,a non-specific hydrolase can catalyze GTP hydrolysis to produce guanosine diphosphate?GDP?with low mimic enzyme activity and guanosine monophosphate?GMP?and guanosine?G?without the catalytic activity under alkaline conditions.Based on this cascade reaction,a highly sensitive colorimetric sensing platform was constructed for ALP sensing.In addition to being used as a biomarker for clinical diagnosis,ALP is widely used in enzyme-linked immunosorbent assays?ELISA?as a signal reporter due to due to its high catalytic activity,broad substrate specificity and commercial availability.Then,as a proof of concept,ALP-labelled enzyme-linked immunosorbent assay for alpha fetoprotein?AFP?was designed coupling with the biocatalytic activity of GTP.The proposed immunoassay has been applied for AFP level detection in serum samples with satisfied results.5.Group effect on the peroxidase-like activity of nucleotidesNucleotides,the basic units of biological macromolecules nucleic acid and deoxynucleic acid,are composed of three subunit molecules:nucleobase?purine base or pyrimidine base?,sugar?ribose or deoxyribose?and at least one phosphate group and play a fundamental role in cell metabolism and regulation.We investigated the peroxidase activity of ATP,GTP and its nucleotide analogues?CTP,UTP,dATP,dGTP,dCTP,dUTP,dTTP,GDP,GMP,G?.The results demonstrated that the peroxidase of GTP and other nucleotides depended on the type of nucleobase and the number of phosphate group,while the glycosyl group had little effect on its catalytic performance.The order of their catalytic activities was:GTP>ATP>UTP>CTP,GTP>GDP>GMP?G?inactive?,dGTP>dTTP?dATP>dUTP>dCTP,dGTP?GTP,dATP?ATP,dCTP?CTP,dUTP?UTP,which was consistent with the results of the activation energy calculated by Arrhenius formula.Electron spin resonance?ESR?experiments identified the formation of·OH radical via H₂O₂decomposition during the catalytic process.Enzyme kinetic analysis and quantum theoretical calculation indicated that nucleobase act as binding sites of substrate H2O2.