Effects of inulin with different polymerization degrees on the structural and gelation properties of potato protein.

This study investigated the effect of inulin with different polymerization degrees (DP), including L-inulin (DP 2-6), M-inulin (DP 10-23) and H-inulin (DP 23-46), on the structural and gelation properties of potato protein isolate (PPI). Results revealed that textural properties (hardness, cohesiveness, springiness and chewiness) and water-holding capacity (WHC) of PPI-inulin composite gels were positively correlated with the inulin DP and addition content at 0-1.5% (w/v), but deteriorated at 2% due to phase separation. The addition of 1.5% H-inulin showed the most significant increment effects on the WHC (18.65%) and hardness (2.84 N) of PPI gel. Furthermore, M-/H-inulin were more effective in increasing the whiteness and surface hydrophobicity, as well as in strengthening hydrogen bonds and hydrophobic interactions than L-inulin. Fourier transform infrared spectroscopy analysis and microstructural observation indicated that inulin with higher DP promoted more generation of ß-sheet structures, and leading to the formation of stronger and finer network structures.

Cross-layer transmission realized by light-emitting memristor for constructing ultra-deep neural network with transfer learning ability.

Deep neural networks have revolutionized several domains, including autonomous driving, cancer detection, and drug design, and are the foundation for massive artificial intelligence models. However, hardware neural network reports still mainly focus on shallow networks (2 to 5 layers). Implementing deep neural networks in hardware is challenging due to the layer-by-layer structure, resulting in long training times, signal interference, and low accuracy due to gradient explosion/vanishing. Here, we utilize negative ultraviolet photoconductive light-emitting memristors with intrinsic parallelism and hardware-software co-design to achieve electrical information's optical cross-layer transmission. We propose a hybrid ultra-deep photoelectric neural network and an ultra-deep super-resolution reconstruction neural network using light-emitting memristors and cross-layer block, expanding the networks to 54 and 135 layers, respectively. Further, two networks enable transfer learning, approaching or surpassing software-designed networks in multi-dataset recognition and high-resolution restoration tasks. These proposed strategies show great potential for high-precision multifunctional hardware neural networks and edge artificial intelligence.

Effect of High-Hydrostatic-Pressure Treatment on the Physicochemical Properties of Kafirin.

The kafirin derived from Jin Nuo 3 sorghum underwent a high-hydrostatic-pressure (HHP) treatment of 100, 300, and 600 MPa for 10 min to investigate alterations in its physicochemical attributes. The findings exhibited a reduction in protein solubility, declining from 83% to 62%, consequent to the application of the HHP treatment. However, this treatment did not lead to subunit-specific aggregation. The absorption intensity of UV light diminished, and the peak fluorescence absorption wavelength exhibited a shift from 342 nm to 344 nm, indicating an increased polarity within the amino acid microenvironment. In an aqueous solution, the specific surface area expanded from 294.2 m2/kg to 304.5 m2/kg, while the average particle-size value in a 70% ethanol solution rose to 26.3 nm. Conversely, the zeta-potential value decreased from 3.4 mV to 1.3 mV, suggesting a propensity for aggregation in ethanol solutions. A notable rise in the intermolecular ß-sheet content to 21.06% was observed, along with a shift in the peak denaturation temperature from 76.33 °C to 86.33 °C. Additionally, the content of disulfide bonds increased to 14.5 µmol/g. Collectively, the application of the HHP treatment not only enhanced the thermal stability but also induced a more ordered secondary structure within the kafirin.

Effect of Freezing on Soybean Protein Solution.

To investigate the impact of frozen storage conditions on the physicochemical properties of soybean protein and explore the underlying mechanisms, this study focused on soybean isolate (SPI), ß-soybean companion globulin (7S), and soybean globulin (11S). The protein solutions were prepared at a concentration of 2% and subjected to freezing for 1 and 5 days. Subsequently, the protein content, physicochemical properties, secondary structure, sulfhydryl content, and chemical interaction forces were assessed and analyzed using UV spectrophotometry, Zeta potential measurements, SDS-PAGE, Fourier infrared spectroscopy, and endogenous fluorescence photoemission spectroscopy. The obtained results revealed that the solubility and total sulfhydryl content of SPI, 7S, and 11S exhibited a decreasing trend with prolonged freezing time. Among them, 11S demonstrated the largest decrease in solubility and total sulfhydryl content, followed by SPI, and 7S the least. During freezing, the aromatic amino acids of SPI, 7S, and 11S molecules were exposed, leading to increased hydrophobicity, protein aggregation, and particle size enlargement, and the structure of the protein changed from disordered structure to ordered structure. After freezing, the polarity of the microenvironment of SPI, 7S, and 11S increased, and their maximum fluorescence emission wavelengths were red-shifted. Notably, the largest red shift of SPI was from 332 nm to 335 nm. As freezing time increased, the contribution of hydrogen bonding increased, while the contribution of hydrophobic interactions decreased. This indicates that freezing affects the hydrophobic interactions, hydrogen bonding, and other chemical forces of the protein. The growth of ice crystals leads to the unfolding of protein molecular chains, exposure of internal hydrophobic groups, enhancement of hydrophobicity, and alters the secondary structure of the protein.

Hydrophilic and hydrophobic deep eutectic solvent-based extraction to determine parathion in cereals by digital image colorimetry integrated with smartphones.

To determine parathion in cereals, hydrophilic and hydrophobic deep eutectic solvents (DESs) were used by digital image colorimetry with smartphones. In the solid-liquid extraction part, hydrophilic DESs were used as extractants to extract parathion from cereals. In the liquid-liquid microextraction part, hydrophobic DESs dissociated into terpineol and tetrabutylammonium bromide in situ. The dissociated hydrophilic tetrabutylammonium ions reacted with parathion extracted in hydrophilic DESs under alkaline conditions to produce a yellow product, which was extracted and concentrated by dispersed organic phase terpinol. Digital image colorimetry integrated with the use of a smartphone was used for quantitative analysis. The limit of detection and quantification were 0.003 mg kg-1 and 0.01 mg kg-1, respectively. The recoveries for parathion were 94.8-106.2% with a relative standard deviation less than 3.6%. The proposed method was applied to analyze parathion in cereal samples: the method has the potential to be applied to pesticide residue analysis in food products.

Dispersive liquid‒liquid microextraction combined with enzyme-linked immunosorbent assay for the analysis of chlorpyrifos in cereal samples.

In this paper, an analysis method for chlorpyrifos (CPF) in cereal samples was proposed using dispersive liquid‒liquid microextraction combined with an enzyme-linked immunosorbent assay. In the dispersive liquid‒liquid microextraction, deep eutectic solvents and fatty acids were used as solvents to extract, purify, and concentrate CPF in cereals. In the enzyme-linked immunosorbent assay, gold nanoparticles were utilized to enrich and conjugate more antibodies and horseradish peroxidase, while magnetic beads were used as solid supports to amplify the signal and shorten the detection time of CPF. The linearity range was 0.002-1 µg kg-1, and the limit of detection was 0.0006 µg kg-1. The extraction recoveries were 86.7-99.9% with a relative standard deviation of less than 7.0%. The proposed method was successfully used to analyze CPF in cereal samples (rice, wheat, maize, and millet) and has prospects for the pretreatment and detection of CPF residues in other food samples.

Protective Effect of Foxtail Millet Protein Hydrolysate on Ethanol and Pyloric Ligation-Induced Gastric Ulcers in Mice.

Foxtail millet has been traditionally considered to possess gastroprotective effects, but studies evaluating its use as a treatment for gastric ulcers are lacking. Here, we assessed the antiulcer effects of foxtail millet protein hydrolysate (FPH) and explored its mechanism by using blocking agents. In a mouse model of ethanol-induced gastric ulcers, pretreatment with FPH reduced the ulcerative lesion index, downregulated the expression of inflammatory cytokines in the gastric tissue, increased the activity of antioxidant enzymes, and improved the oxidative status. FPH increased constitutive the activity of nitric oxide synthase (cNOS), NO levels, and mucin expression in gastric mucosa, and inhibited the activation of the ET-1/PI3K/Akt pathway. In a mouse model of pyloric ligation-induced gastric ulcers, FPH inhibited gastric acid secretion and decreased the activity of gastric protease. Pretreatment of mice with the sulfhydryl blocker NEM and the NO synthesis inhibitor L-NAME abolished the gastroprotective effect of FPH, but not the KATP channel blocker glibenclamide and the PGE2 synthesis blocker indomethacin. Among the peptides identified in FPH, 10 peptides were predicted to have regulatory effects on the gastric mucosa, and the key sequences were GP and PG. The results confirmed the gastroprotective effect of FPH and revealed that its mechanism was through the regulation of gastric mucosal mucus and NO synthesis. This study supports the health effects of a millet-enriched diet and provides a basis for millet protein as a functional food to improve gastric ulcers and its related oxidative stress.

Tartary Buckwheat (Fagopyrum tataricum) Ameliorates Lipid Metabolism Disorders and Gut Microbiota Dysbiosis in High-Fat Diet-Fed Mice.

Jinqiao II, a newly cultivated variety of tartary buckwheat (Fagopyrum tataricum), has been reported to exhibit a higher yield and elevated levels of functional compounds compared to traditional native breeds. We aimed to investigate the potential of Jinqiao II tartary buckwheat to alleviate lipid metabolism disorders by detecting serum biochemistry, pathological symptoms, gene expression profiling, and gut microbial diversity. C57BL/6J mice were provided with either a normal diet; a high-fat diet (HFD); or HFD containing 5%, 10%, and 20% buckwheat for 8 weeks. Our results indicate that Jinqiao II tartary buckwheat attenuated HFD-induced hyperlipidemia, fat accumulation, hepatic damage, endotoxemia, inflammation, abnormal hormonal profiles, and differential lipid-metabolism-related gene expression at mRNA and protein levels in response to the dosages, and high-dose tartary buckwheat exerted optimal outcomes. Gut microbiota sequencing also revealed that the Jinqiao II tartary buckwheat elevated the level of microbial diversity and the abundance of advantageous microbes (Alistipes and Alloprevotella), lowered the abundance of opportunistic pathogens (Ruminococcaceae, Blautia, Ruminiclostridium, Bilophila, and Oscillibacter), and altered the intestinal microbiota structure in mice fed with HFD. These findings suggest that Jinqiao II tartary buckwheat might serve as a competitive candidate in the development of functional food to prevent lipid metabolic abnormalities.

Bioinspired Artificial Motion Sensory System for Rotation Recognition and Rapid Self-Protection.

As one of the most common synergies between the exteroceptors and proprioceptors, the synergy between visual and vestibule enables the human brain to judge the state of human motion, which is essential for motion recognition and human self-protection. Hence, in this work, an artificial motion sensory system (AMSS) based on artificial vestibule and visual is developed, which consists of a tribo-nanogenerator (TENG) as a vestibule that can sense rotation and synaptic transistor array as retina. The principle of temporal congruency has been successfully realized by multisensory input. In addition, pattern recognition results show that the accuracy of multisensory integration is more than 15% higher than that of single sensory. Moreover, due to the rotation recognition and visual recognition functions of AMSS, we realized multimodal information recognition including angles and numbers in the spiking correlated neural network (SCNN), and the accuracy rate reached 89.82%. Besides, the rapid self-protection of a human was successfully realized by AMSS in the case of simulated amusement rides, and the reaction time of multiple motion sensory integration is only one-third of that of a single vestibule. The development of AMSS based on the synergy of simulated vision and vestibule will show great potential in neural robot, artificial limbs, and soft electronics.

Determination of methomyl in grain using deep eutectic solvent-based extraction combined with fluorescence-based enzyme inhibition assays.

A deep eutectic solvent (DES)-based extraction method is established to facilitate the determination of methomyl in grain via enzyme inhibition fluorescence. The environmentally-friendly DES was synthesized from proline and ethylene glycol and used as a green replacement for traditional extraction solvents that are generally toxic. The DES was added to grain samples and vortex extraction of methomyl, the supernatant was then collected for fluorescence detection. Biomass carbon quantum dots (CQDs) synthesized from millet were used as fluorescent probes. Acetylcholinesterase catalyzes the hydrolysis of acetylthiocholine iodide to thiocholine. The positively-charged thiocholine interacts electrostatically with the negatively-charged quantum dots resulting in the quenching of their fluorescent emission. The pesticide extract solution blocks the enzyme activity and thus recovers the fluorescent from the quantum dots. The fluorescence response was correlated with the amount of methomyl residue in the grain over the range 0.01 to 5 mg kg-1. The limit of detection was found to be 0.003 mg kg-1, and the limit of quantification 0.01 mg kg-1. Recoveries of 86.5% to 107.8% were obtained using real samples, including millet, rice, wheat, and barley, with a relative standard deviation of less than 3.8%. The method is efficient and convenient and has good application prospects for extracting and detecting pesticides in grain samples.

Micronization Effects on Structural, Functional, and Antioxidant Properties of Wheat Bran.

To explore the effect of micronization on the structural, functional, and antioxidant properties of wheat bran, wheat bran with mean particle size (D50) of 46.08, 34.29, 26.51, 26.35, and 26.05 µm were prepared by using an ultrafine pulverizer under different rolling frequencies (0, 6, 9, 12, and 15 times). The main chemical components and structural, functional, and antioxidant properties of the wheat bran were compared and a correlation analysis was conducted. As the D50 of the wheat bran decreased from 46.08 µm to 26.05 µm, the micromorphology exhibited the destruction of the bundle structure, which is formed by starch and fiber, during which the starch particles peeled off, the fiber fragments destructed, and some of the slim fiber fragments attached to the surfaces of the starch granules. According to the X-ray diffraction pattern, part of the crystalline structure was transformed into an amorphous structure and the crystallization index decreased from 13.08% to 3.95%. According to the near-infrared spectrum, more active groups, such as the hydroxyl group, were exposed; however, no new groups were generated. These structural changes accordingly caused changes in the chemical components, functional properties, and antioxidant properties of the wheat bran. Specifically, the protein, total phenols, total flavonoids, and fatty acid content increased by 6.72%, 23.47%, 19.07%, and 172.88%, respectively. The lipase activity, antioxidant activity in vitro (DPPH• scavenging activity, ABTS+• scavenging activity, and ferric reducing antioxidant power), and the water-holding, cholesterol-adsorption, sodium nitrite-adsorption, and cation-exchange capacities, were enhanced to some extent. The oil-holding capacity decreased from 3.01 g/g to 1.32 g/g. The swelling capacity decreased first and then increased and the swelling capacity of the wheat bran with a D50 of 34.29 µm was the lowest (3.62 mL/g). Therefore, the micronization could be used as a pretreatment method to improve the functional and antioxidant properties of wheat bran; however, the optimal particle size of wheat bran is based on the function of the product.

Wireless signal modulation identification method based on RF I/Q data distribution.

Electromagnetic spectrum detection is the basis of the next generation wireless communication technology. Wireless signal identification is an important part of electromagnetic spectrum detection and management activities. This paper proposes to extract the distribution features of different modulated signals from the signal I/Q data. A two-dimensional gradient matrix is used to describe the characteristics of the signal classification. The minimum gradient cumulative distance (GCD) estimate between the sample and the model is used as the decision criterion for the signal classification. According to the result of the confusion matrix, the weight of the model is adjusted. Experiments show that the recognition rate of the modulated signal mentioned in this paper can reach 82.75%. The I/Q data sample was extracted under actual engineering conditions involving random noise, and the recognition rate dropped to approximately 79%. Based on the initial model gradient matrix, a reasonable algorithm is set to adjust the weight of the model, which can effectively improve the recognition rate of the modulated signal.

Analysis of pyrethroids in cereals by HPLC with a deep eutectic solvent-based dispersive liquid-liquid microextraction with solidification of floating organic droplets.

This work presents a novel and green analytical procedure involving a deep eutectic solvent-based dispersive liquid-liquid microextraction with solidification of floating organic droplets (DES-DLLME-SFOD) followed by HPLC to measure three pyrethroids (bifenthrin, ß-cypermethrin, and deltamethrin) in cereal samples. Firstly, a low-density hydrophobic DES was synthesized from thymol and octanoic acid in the molar ratio of 1/4 and this was applied as a green extraction solvent in the DLLME procedure to avoid the use of a toxic extractant. After centrifugation and placing it on an ice bath, it is transformed into a solid phase on the top of the sample solution to reduce the loss of extractant, conducive to convenient collection thereafter. This procedure required the optimal conditions (including the type, proportion, and amount of DES as the extractant, the volume of the dispersant acetonitrile, the amount of salt, and the pH value) to be evaluated. Under optimized variates, the proposed method provided good linearity with a correlation coefficient greater than 0.997 and limits of quantification within the range of 6.6-8.9 µg kg-1. The recoveries of pyrethroids in corn, wheat, barley, and oats were 75.6-87.2%, and the relative standard deviation was less than 3.6%. The method, therefore, offers a green, efficient, and convenient approach for the determination of pesticides in cereals.

Digital image colorimetry detection of carbaryl in food samples based on liquid phase microextraction coupled with a microfluidic thread-based analytical device.

This research used a digital image colorimetry (DIC) method to detect carbaryl in food samples using effervescence-assisted liquid phase microextraction based on solidification of switchable hydrophilicity solvent combined with a microfluidic thread-based analytical device (EA-LPME-SSHS-µTAD). 1-naphthol, the hydrolysate of carbaryl, was extracted into octanoic acid by the adjustment of pH values of the sample solution and separated through solidification in an ice bath. Then 1-naphthol contained in the extracted solution was coupled with 4-methoxybenzenediazonlum tetrafluoroborate (MBDF) fixed on the µTAD to produce tangerine compounds. The inherent colour variation was captured by a smartphone and processed to calculate the intensity (I). Under the optimal conditions, the limit of quantification was within 0.020-0.027 mg kg-1. The recovery was varied in the range from 92.3% to 105.9% with a relative standard deviation (RSD) below 5%. The developed method provides an alternative strategy to extract and detect pesticides for food samples.

Characterization of aldehydes and hydroxy acids as the main contribution to the traditional Chinese rose vinegar by flavor and taste analyses.

The volatile aroma compounds of traditional Chinese rose vinegar were identified by headspace solid-phase micro extraction gas chromatography-mass spectrometry (HS-SPME-GC-MS) and GC-MS-olfactometry (GC-MS-O), and the metabolites were identified by silylation-GC-MS in this study. A total of 48 and 76 kinds of flavors and metabolites, respectively were detected in this study. Quantitative analysis showed that aldehydes and acids were present in relatively high amounts. Furthermore, the data colleted by the calculated odor activity values (OAVs) suggested that aldehydes are likely to contribute greatly to the aroma of traditional Chinese rose vinegar, especially, nonanal (OAV: 133, rose), 3-methyl-butanal (OAV: 57, apple-like), decanal (OAV: 23, orange peel), heptanal (OAV: 17, fruity), and dodecanal (OAV: 4-9, violet scents). Moreover, among the detected nonvolatile acids, 14 kinds of hydroxy acids, such as lactic acid, citric acid, 3-phenyllactic acid (PLA) and d-gluconic acid were detected in rose vinegar. The acids provide a well buffer system, not only greatly reduce the irritation of acetic acid, but also improve the flavor of rose vinegar. This study suggests that the fragrance and sour notes in rose vinegar are from aldehydes and hydroxy acids.

Evaporation-assisted dispersive liquid-liquid microextraction based on the solidification of floating organic droplets for the determination of triazole fungicides in water samples by high-performance liquid chromatography.

A simple, rapid, and effective analytical procedure for determining three triazole fungicides (myclobutanil, epoxiconazole, and tebuconazole) in water samples is developed by high-performance liquid chromatography-diode array detection after evaporation-assisted dispersive liquid-liquid microextraction based on the solidification of floating organic droplets. The extraction procedure involves the sequential addition of the extraction solvent 1-dodecanol (low density), volatile solvent dichloromethane (high density), and calcium oxide to the aqueous sample (the latter reacting exothermically). The CaO reaction can promote the volatilization of the dichloromethane which disperses the 1-dodecanol as fine droplets in the aqueous sample due to the bubbles generated. Therefore, a dispersive solvent is not required. Then, the floating 1-dodecanol is solidified using an ice bath for easy separation from the sample. The variables (the volumes of extraction and volatile solvents, amounts of calcium oxide and sodium chloride, pH values, and extraction time) in the extraction procedure are further optimized. Under optimized conditions, the linearity ranges are 0.05-5 µg mL-1 with correlation coefficients greater than 0.99. The limits of detection and quantification are 0.0051-0.0090 µg mL-1 and 0.0169-0.0299 µg mL-1, respectively. The recoveries of myclobutanil, epoxiconazole, and tebuconazole in tap, reservoir, and river water range between 77.6% and 104.4% with relative standard deviations ranging from 0.6% to 7.8%. Hence, the method was reliable for analysis of myclobutanil, epoxiconazole, and tebuconazole in water samples.

Design, synthesis and biological evaluation of novel 1-phenyl phenanthridin-6(5H)-one derivatives as anti-tumor agents targeting TOPK.

T-lymphokine-activated killer cell-originated protein kinase (TOPK) is a serine-threonine mitogen-activated protein kinase that is highly expressed in many types of human cancer. Due to its important role in cancer progression, TOPK is becoming an attractive target in chemotherapeutic drug design. In this study, a series of 1-phenyl phenanthridin-6(5H)-one derivatives have been identified as a novel chemical class of TOPK inhibitors. Some of them displayed very potent anti-cancer activity with IC50s less than 100 nM, superior than reference compound OTS964. The most potent compound, 9g suppressed the growth of cancer cells by apoptosis and specifically inhibited the activities of TOPK. Oral administration of 9g effectively suppressed tumor growth with TGI >79.7% in colorectal cancer xenograft models, demonstrating superior efficacy compared to OTS964. Pharmacokinetic studies reveal its good oral bioavailability. Our findings therefore show that 9g is a specific inhibitor of TOPK both in vitro and in vivo that may be further developed as a potential therapeutic agent against colorectal cancer.

Effervescent-assisted dispersive liquid-liquid microextraction based on the solidification of floating organic droplets for the determination of fungicides in vinegar and juice.

A method of effervescent-assisted dispersive liquid-liquid microextraction based on the solidification of a floating organic droplet is reported. This approach was used to measure the fungicides myclobutanil, tebuconazole and epoxiconazole in vinegar and juice. Acidic vinegar and juice reacted with the carbonate to produce effervescence in situ, which then promoted contact of the sample with the extraction solvent. The 1-dodecanol extraction solvent helped solidify the floating organic droplets and could be fully dispersed by effervescence. The extraction solvent, salt, carbonate, and extraction time were optimised. The optimal conditions were 200 µL of 1-dodecanol, 100 mg of sodium chloride, 50 mg of sodium bicarbonate, and 30 s of extraction time. The proposed method has good linearity between 10 and 1000 ng mL-1. Recoveries were between 70.4% and 103.1% in different vinegar and juice samples. This method was successfully used to measure fungicides in vinegar and juice. This system is simple, fast and environmentally friendly.

Yield and textural properties of tofu as affected by soymilk coagulation prepared by a high-temperature pressure cooking process.

The cooking of raw soymilk is a necessary procedure prior to the production of tofu. The effects of the high-temperature pressure cooking (HTPC) and traditional cooking methods on the yield and textural properties of tofu products were investigated. Results showed that when HTPC was applied, the content of protein particles increased, thereby contributing to the formation of a dense network of tofu gel. Thus, significant improvement of textural properties, including hardness, chewiness and springiness, was observed. Moreover, HTPC contributes to the change in the composition of the particulate protein, whereas the proportion of ß-conglycinin in the non-particulate protein increased. The start and end points of the protein coagulation induced by Ca(2+) moved backward, and slowed the coagulation process, which was conducive to the incorporation of water or dry matter into the gel.

The kinetic role of carboxylate residues in the proximity of the trinuclear centre in the O2 reactivity of CotA-laccase.

Multicopper oxidases catalyze the four-electron reduction of dioxygen to water without the release of any reactive oxygen intermediate species. The role of carboxylate residue Asp116 located at the exit channel for water molecules of CotA-laccase has been investigated by site-saturation mutagenesis. A total of 300 clones was picked and screened for activity. Five variant enzymes, D116E, D116A, D116N, D116T and D116L, were selected for further characterisation. Spectroscopic analysis revealed only small perturbations in the geometry of the catalytic Cu sites of variants. However, a severe drop in turnover numbers (k(cat)) and downshifts by approximately 1-2 units of the optimal pH were observed for the oxidation of substrates, as compared with the wild type. The kinetics of formation and decay of peroxide intermediate (PI) was studied in type 1 depleted (T1D) CotA-laccase and in T1D-D116 or T1D-E498 mutants, previously shown to be involved in the mechanism of dioxygen reduction. It is noteworthy that CotA shows 10 times lower rates of PI formation and 10(3) higher PI decay rates as compared with other studied multicopper oxidases. The generation of PI is pH independent and mostly unaffected by the D116 or E498 mutations. In contrast, the decay of PI is markedly compromised by the replacement of D116 or E498 with non-carboxylate residues. The E498 residue appears to be the main protonable species for acceleration of PI decay at low pH. The D116 residue seems to be essential in the modulation of E498 protonation and in assisting protons to hydroxyl groups bound to the T2 Cu.