High Isoelectric Point Research Articles

ZnO nanostructures in enzyme biosensors

Biosensing has developed tremendously since it was demonstrated by Leland C. Clark Jr. in 1962. ZnO nanomaterials are attractive candidates for fabricating biosensors, because of their diverse range of nanostructures, high electron mobility, chemical stability, electrochemical activity, high isoelectric points which promote enzyme adsorption, biocompatibility, and piezoelectric properties. This review covers ZnO nanostructures applied in enzyme biosensors, in the light of electrochemical transduction and field effect transduction. Different assembly processes and immobilization methods have been used to load enzymes into various ZnO nanostructures, providing enzymes with favorable micro-environments and enhancing their sensing performance. We briefly describe recent trends in ZnO syntheses, and the analytical performance of the fabricated biosensors, summarize the advantages of using ZnO nanostructures in biosensors, and conclude with future challenges and prospects.

Nanoporous CuO layer modified Cu electrode for high performance enzymatic and non-enzymatic glucose sensing

Nanoporous CuO layer on Cu foil with a thick Cu2O interlayer is synthesized via post annealing of previously fabricated Cu(OH)2 nanowires at 500 °C under an oxygen flow. The formation of the thick sandwiched Cu2O layer is realized through the outward diffusion of Cu ions and subsequent oxidation. An O2 pressure above the dissociation pressure of CuO is used to form a CuO layer at the outer surface of the structure, thus realizing a low cost structure having a porous and high isoelectric point layer. The Cu/Cu2O/CuO structure is used as an efficient electrode for glucose sensing. Sensitivities of at 0.8 V versus Ag/AgCl and 1066 μA mM–1 cm–2 at 0.6 V versus Ag/AgCl are achieved in an enzymatic and non-enzymatic glucose sensing schemes, respectively. The improved electrochemical sensing ability might be attributed to the efficient electrocatalytic reaction on the high crystal quality CuO layer and the porous structure.

Glycoproteomics: identifying the glycosylation of prostate specific antigen at normal and high isoelectric points by LC-MS/MS.

Prostate specific antigen (PSA) is currently used as a biomarker to diagnose prostate cancer. PSA testing has been widely used to detect and screen prostate cancer. However, in the diagnostic gray zone, the PSA test does not clearly distinguish between benign prostate hypertrophy and prostate cancer due to their overlap. To develop more specific and sensitive candidate biomarkers for prostate cancer, an in-depth understanding of the biochemical characteristics of PSA (such as glycosylation) is needed. PSA has a single glycosylation site at Asn69, with glycans constituting approximately 8% of the protein by weight. Here, we report the comprehensive identification and quantitation of N-glycans from two PSA isoforms using LC–MS/MS. There were 56 N-glycans associated with PSA, whereas 57 N-glycans were observed in the case of the PSA-high isoelectric point (pI) isoform (PSAH). Three sulfated/phosphorylated glycopeptides were detected, the identification of which was supported by tandem MS data. One of these sulfated/phosphorylated N-glycans, HexNAc5Hex4dHex1s/p1 was identified in both PSA and PSAH at relative intensities of 0.52 and 0.28%, respectively. Quantitatively, the variations were monitored between these two isoforms. Because we were one of the laboratories participating in the 2012 ABRF Glycoprotein Research Group (gPRG) study, those results were compared to that presented in this study. Our qualitative and quantitative results summarized here were comparable to those that were summarized in the interlaboratory study.

Adsorption of enzyme onto lignins of liquid hot water pretreated hardwoods.

The adsorption of cellulase enzymes onto lignin is shown to be non-productive and therefore reduces enzymatic hydrolysis of liquid hot water pretreated cellulose. Among the enzyme components of Trichoderma reesei cellulase cocktail, β-glucosidase showed the strongest adsorption onto lignin. Only 2-18% of the initial β-glucosidase activity remained in the supernatant while 50-60% of cellobiohydrolase and endoglucanase activities were recovered after incubation with lignin. By increasing the pH to 5.5 and adding NaCl to a 200 mM, the free enzymes in the supernatant were increased but hydrolysis was not enhanced since optimal pH for enzymatic hydrolysis is at 4.8. Electrostatic interactions contributed to enzyme adsorption and their effect was most pronounced for T. reesei β-glucosidase which had high molecular weights (78-94 kDa) and high isoelectric points (pI 5.7-6.4). Since the enzyme components which are required to synergistically hydrolyze cellulose have different profiles (molecular weight, hydrophobicity and pI), they exhibit different adsorption behaviors with lignin, and thereby change the ratio of enzyme activities needed for synergism during cellulose hydrolysis. β-glucosidase from Aspergillus niger exhibits less adsorption than β-glucosidase from T. reesei. Supplemental addition of A. niger β-glucosidase to the enzyme mixture increases hydrolysis of pretreated hardwood by a factor of two. The analysis presented in this paper shows that lignins with higher guaiacyl content adsorb more cellulase enzymes, particularly β-glucosidase, and that adsorption of β-glucosidase onto lignin indirectly suppresses enzymatic hydrolysis of cellulose in pretreated hardwoods due to decreased hydrolysis of cellobiose which in turn accumulates and inhibits CBH.

Functionalization of Magnetic Multiwalled Carbon Nanotubes with Mixed Surfactants for Enhancing Protein Adsorption

Magnetic multiwalled carbon nanotubes (M-MWNTs) have been functionalized with mixed surfactants consisting of a sugar-based surfactant and an ionic surfactant. The synergistic effect of the two surfactants results in more surfactants adsorbed and the formation of a more disordered structure of assembled surfactants. These two aspects facilitate simultaneous hydrogen bonding and electrostatic interactions with proteins. It has been demonstrated that the M-MWNTs functionalized with mixed surfactants adsorbed more proteins than the M-MWNTs functionalized with single surfactants. The M-MWNTs functionalized with mixed surfactants have been tested for the adsorption of two proteins, bovine serum albumin (BSA) with a low isoelectric point and lysozyme with a high isoelectric point, and similar results have been obtained. Hence, carbon nanotubes functionalized with mixed surfactants have a wide range of potential applications for protein adsorption.

PDMS 채널 내부에 성장된 산화아연 나노막대를 이용한 H7N9 인플루엔자 바이러스 전기화학 면역센서

In this study, we propose an immunosensor using zinc oxide nanorods (NRs) inside PDMS channel for detecting the influenza A virus subtype H7N9. ZnO with high isoelectric point (IEP, ~9.5) makes it suitable for immobilizing proteins with low IEP. In this proposed H7N9 immunosensor structure ZnO NRs were grown on the PDMS channel inner surface to immobilize H7N9 capture antibody. A sandwich enzyme-linked immunosorbent assay (ELISA) method with was used 3,3',5,5' tetramethylbenzidine (TMB) for detecting H7N9 influenza virus. The immunosensor was evaluated by amperometry at various H7N9 influenza antigen concentrations (1 pg/ml - 1 ng/ml). The redox peak voltage and current were measured by amperometry with ZnO NWs and without ZnO NWs inside PDMS channel. The measurement results of the H7N9 immunosensor showed that oxidation peak current of TMB at 0.25 V logarithmically increased from 2.3 to 3.8 uA as the H7N9 influenza antigen concentration changed from 1 pg/ml to 1 ng/ml. And then we demonstrated that ZnO NRs inside PDMS channel can improve the sensitivity of immunosensor to compare non-ZnO NRs inside PDMS channel.

Electrospun Porous ZnO Nanofibers for Glucose Biosensors

Porous 1 dimensional (1D) ZnO nanofibers were synthesized by electrospinning technology and sequent annealing process. The ZnO nanofibers have many nanopores in it and connect to each other to form a porous film. The diameters of the nanofibers are about 100 nm, and their lengths are so long as tens of millimeters. Owing to the porous nanofibers based matrix has 1D channels and high isoelectric point, the prepared ZnO porous film is wonderful platform to immobilize glucose oxidase enzyme for glucose biosensing. The as-fabricated biosensor exhibited high sensitivity (69 μA/mMcm-2), fast response (3 s) and low detection limit (10 μM), due to the nanomaterial with large surface-to-volume ratio for enzyme immobilization and excellent electrical properties. The biosensors can be fabricated controllably and repeatedly. These excellent results indicate the biosensors are attractive for application.

Effects of chlorate on the sulfation process of Trypanosoma cruzi glycoconjugates. Implication of parasite sulfates in cellular invasion

Sulfation, a post-translational modification which plays a key role in various biological processes, is inhibited by competition with chlorate. In Trypanosoma cruzi, the agent of Chagas’ disease, sulfated structures have been described as part of glycolipids and we have reported sulfated high-mannose type oligosaccharides in the C-T domain of the cruzipain (Cz) glycoprotein. However, sulfation pathways have not been described yet in this parasite. Herein, we studied the effect of chlorate treatment on T. cruzi with the aim to gain some knowledge about sulfation metabolism and the role of sulfated molecules in this parasite. In chlorate-treated epimastigotes, immunoblotting with anti-sulfates enriched Cz IgGs (AS-enriched IgGs) showed Cz undersulfation. Accordingly, a Cz mobility shift toward higher isoelectric points was observed in 2D-PAGE probed with anti-Cz antibodies. Ultrastructural membrane abnormalities and a significant decrease of dark lipid reservosomes were shown by electron microscopy and a significant decrease in sulfatide levels was confirmed by TLC/UV-MALDI-TOF-MS analysis. Altogether, these results suggest T. cruzi sulfation occurs via PAPS. Sulfated epitopes in trypomastigote and amastigote forms were evidenced using AS-enriched IgGs by immunoblotting. Their presence on trypomastigotes surface was demonstrated by flow cytometry and IF with Cz/dCz specific antibodies. Interestingly, the percentage of infected cardiac HL-1 cells decreased 40% when using chlorate-treated trypomastigotes, suggesting sulfates are involved in the invasion process. The same effect was observed when cells were pre-incubated with dCz, dC-T or an anti-high mannose receptor (HMR) antibody, suggesting Cz sulfates and HMR are also involved in the infection process by T. cruzi.

PH-Responsive poly(itaconic acid-co-N-vinylpyrrolidone) hydrogels with reduced ionic strength loading solutions offer improved oral delivery potential for high isoelectric point-exhibiting therapeutic proteins.

pH-Responsive hydrogels comprised of itaconic acid copolymerized with N-vinylpyrrolidone (P(IA-co-NVP)) were synthesized and tested as carriers for the oral delivery of high isoelectric point (pI) exhibiting therapeutic proteins. Swelling studies show that P(IA-co-NVP) hydrogels exhibit significantly greater and faster pH-responsive swelling than previously studied methacrylic acid-based hydrogels, achieving up to 68% greater equilibrium swelling and 10.4 times greater swelling in time-limited experiments. Using salmon calcitonin as a model high pI protein therapeutic, we show that P(IA-co-NVP) hydrogels exhibit significantly greater delivery potential than methacrylic acid-based hydrogels. Additionally, we show that utilizing a lower ionic strength solution during drug loading significantly improves drug delivery potential for high pI therapeutics. By using a 1.5mM PBS buffer rather than the standard 150 mM PBS buffer during loading, up to 83 times as much calcitonin can be delivered in neutral conditions, with up to a 9.6-fold improvement in percent release. Using P(IA-co-NVP) hydrogel microparticles and a low ionic strength loading solution, up to 48 μg calcitonin/mg hydrogel can be delivered in small intestinal conditions. Based on expected absorption in the small intestine, this is sufficient delivery potential for achieving therapeutic dosage via a single, regularly-sized pill taken daily.

Effect of carboxyl density at the core–shell interface of surface-imprinted magnetic trilayer microspheres on recognition properties of proteins

A novel protein surface-imprinted magnetic trilayer composite microsphere (Fe3O4@HEA@protein-MIPs) was designed and prepared. The objective of this study was to study the effect of carboxyl density at the core–shell interface on recognition performance. The imprinting process was carried out on the surface of Fe3O4@hydroxyethyl acrylate (Fe3O4@HEA) microsphere modified by maleic anhydride (MAH) in the presence of functional monomer acrylamide (AM) and cross-linker N,N′-methylenebisacrylamide (MBA). The results revealed that the adsorption rate, adsorption capacity and recognition capacity of surface-imprinted magnetic microsphere increased with increasing carboxyl density. Meanwhile, the increase of carboxyl density was conducive to improve the imprinting efficiency of the proteins with high isoelectric point (pI>7). Furthermore, the effect of carboxyl at the core–shell interface on imprinting efficiency was restricted by the thickness of imprinted layer, and the critical thickness which related to the size of template protein was obtained by experiments.

Control of Lysozyme Adsorption by pH on Surfaces Modified with Polyampholyte Brushes

The adsorption of lysozyme is difficult to control by pH because of the relatively high isoelectric point of this protein (11.1). In this article, we demonstrate good control of lysozyme adsorption by pH in the range of 4-10 on silicon surfaces through modification with poly(2-(dimethylamino ethyl) methacrylate)-block-poly(methacrylic acid) (PDMAEMA-b-PMAA) diblock copolymer brushes. We show that the thickness of the outer PMAA block (lPMAA) is critical to the adsorption. When lPMAA was less than 10 nm, adsorption increased with increasing pH, and the difference in adsorption between high and low pH increased with lPMAA. The ratio of adsorption at pH 10 and pH 4 reached values as high as 16.4. When lPMAA was more than 10 nm, the adsorption tendency on the PDMAEMA-b-PMAA diblock copolymer brushes was similar to that on PMAA homopolymer brushes. These results indicate that the combination of PDMAEMA and PMAA gives adsorption behavior reflecting the properties of both polymers. However, if the outer PMAA block is thicker than a critical value, then the protein-resistant effect of the inner PDMAEMA block is screened.

The contribution of different alpha-amylase isoenzymes of the commodity grain spring wheat in the formation of falling number values

The participation of various isoenzymes of alpha-amylase in the formation of falling number values of the commodity grain of wheat grown in the Republic of Kazakhstan was investigated. It was found that active isoenzymes alpha-AMY1 and alpha-AMY2 of the embryonic shield were present in the grain with an index over 200. A significant decrease in the falling number depended mainly on the synthesis of alpha-AMY1 and alpha-AMY2 isoenzymes in the aleurone layer. In the grain, isoenzymes with high isoelectric points (p1 > or = 7.3) were found; these isoenzymes belong to alpha-amylase or late maturing or alpha-amylase of practically mature grains. It was discovered that the exogenous hormone (gibberellic acid) induced synthesis of alpha-amylase isoenzymes of scutellum, whole caryopses, and aleurone. It was shown that the impact of exogenous gibberellic acid on the activity and structure of alpha-amylase is reduced in grain with a low falling number.

Recombinant Human Factor IX Produced from Transgenic Porcine Milk

Production of biopharmaceuticals from transgenic animal milk is a cost-effective method for highly complex proteins that cannot be efficiently produced using conventional systems such as microorganisms or animal cells. Yields of recombinant human factor IX (rhFIX) produced from transgenic porcine milk under the control of the bovine α-lactalbumin promoter reached 0.25 mg/mL. The rhFIX protein was purified from transgenic porcine milk using a three-column purification scheme after a precipitation step to remove casein. The purified protein had high specific activity and a low ratio of the active form (FIXa). The purified rhFIX had 11.9 γ-carboxyglutamic acid (Gla) residues/mol protein, which approached full occupancy of the 12 potential sites in the Gla domain. The rhFIX was shown to have a higher isoelectric point and lower sialic acid content than plasma-derived FIX (pdFIX). The rhFIX had the same N-glycosylation sites and phosphorylation sites as pdFIX, but had a higher specific activity. These results suggest that rhFIX produced from porcine milk is physiologically active and they support the use of transgenic animals as bioreactors for industrial scale production in milk.

The Potato Tuber Mitochondrial Proteome

Mitochondria are called the powerhouses of the cell. To better understand the role of mitochondria in maintaining and regulating metabolism in storage tissues, highly purified mitochondria were isolated from dormant potato tubers (Solanum tuberosum 'Folva') and their proteome investigated. Proteins were resolved by one-dimensional gel electrophoresis, and tryptic peptides were extracted from gel slices and analyzed by liquid chromatography-tandem mass spectrometry using an Orbitrap XL. Using four different search programs, a total of 1,060 nonredundant proteins were identified in a quantitative manner using normalized spectral counts including as many as 5-fold more "extreme" proteins (low mass, high isoelectric point, hydrophobic) than previous mitochondrial proteome studies. We estimate that this compendium of proteins represents a high coverage of the potato tuber mitochondrial proteome (possibly as high as 85%). The dynamic range of protein expression spanned 1,800-fold and included nearly all components of the electron transport chain, tricarboxylic acid cycle, and protein import apparatus. Additionally, we identified 71 pentatricopeptide repeat proteins, 29 membrane carriers/transporters, a number of new proteins involved in coenzyme biosynthesis and iron metabolism, the pyruvate dehydrogenase kinase, and a type 2C protein phosphatase that may catalyze the dephosphorylation of the pyruvate dehydrogenase complex. Systematic analysis of prominent posttranslational modifications revealed that more than 50% of the identified proteins harbor at least one modification. The most prominently observed class of posttranslational modifications was oxidative modifications. This study reveals approximately 500 new or previously unconfirmed plant mitochondrial proteins and outlines a facile strategy for unbiased, near-comprehensive identification of mitochondrial proteins and their modified forms.

Enhanced selectivity of hydrogel-based molecularly imprinted polymers (HydroMIPs) following buffer conditioning

We have investigated the effect of buffer solution composition and pH during the preparation, washing and re-loading phases within a family of acrylamide-based molecularly imprinted polymers (MIPs) for bovine haemoglobin (BHb), equine myoglobin (EMb) and bovine catalyse (BCat). We investigated water, phosphate buffer saline (PBS), tris(hydroxymethyl)aminomethane (Tris) buffer and succinate buffer. Throughout the study MIP selectivity was highest for acrylamide, followed by N-hydroxymethylacrylamide, and then N-iso-propylacrylamide MIPs. The selectivity of the MIPs when compared with the NIPs decreased depending on the buffer conditions and pH in the order of Tris>PBS>succinate. The Tris buffer provided optimum imprinting conditions at 50mM and pH 7.4, and MIP selectivities for the imprinting of BHb in polyacrylamide increased from an initial 8:1 to a 128:1 ratio. It was noted that the buffer conditions for the re-loading stage was important for determining MIP selectivity and the buffer conditions for the preparation stage was found to be less critical. We demonstrated that once MIPs are conditioned using Tris or PBS buffers (pH7.4) protein reloading in water should be avoided as negative effects on the MIP's imprinting capability results in low selectivities of 0.8:1. Furthermore, acidifying the pH of the buffer solution below pH 5.9 also has a negative impact on MIP selectivity especially for proteins with high isoelectric points. These buffer conditioning effects have also been successfully demonstrated in terms of MIP efficiency in real biological samples, namely plasma and serum.

Highly linear pH gradients for analyzing monoclonal antibody charge heterogeneity in the alkaline range

Recombinant antibodies with high isoelectric point are frequent since most of them are constructed from the same framework. Classically, cation exchange chromatography is used as a standard method for the determination of antibody charge heterogeneity. In contrast, in this study highly linear pH gradients were achieved by keeping the buffering capacity over the length of the gradient constant. The buffering compounds were selected to be unretained on the column and their respective concentration was adjusted in the start and end buffer of the pH gradient to achieve constant buffering capacity. This helps conserve linearity and stability of the gradient. The method allows quantification of charge variant distribution and the determination of chromatographic isoelectric point. To demonstrate the effectiveness of this novel method, a ProPac WCX-10 column was used to separate isoforms of trastuzumab biosimilar antibodies. Effects of pH gradient linearity and of varying the analytical amount of sample on the separation are shown.

Enhanced catalytic hydrogenation of aqueous bromate over Pd/mesoporous carbon nitride

Pd catalysts supported on mesoporous carbon nitride (MCN) were synthesized and the liquid phase catalytic hydrogenation of bromate was investigated. Characterization results showed that the Pd/MCN catalysts had large BET surface area, high isoelectric point (IEP), ordered mesoporous structure and well dispersed Pd particles in the pores of MCN. Compared with Pd/active carbon (Pd/AC) and Pd/ordered mesoporous carbon (Pd/CMK-3), Pd/MCN displayed higher catalytic activity for bromate reduction, due to its mesoporous structure and high IEP. The bromate hydrogenation process over Pd/MCN followed the Langmuir–Hinshelwood model, reflecting an adsorption controlling reaction mechanism. The pH-catalytic activity linked relationship was related to the adsorption ability of catalysts. Turn over frequencies (TOFs) of the surface Pd sites were found to be dependent on Pd particle size. The coexisting anions suppressed the reduction of bromate. The present results demonstrated that Pd/MCN could be a promising catalyst for the application on bromate hydrogenation.

A Novel Astaxanthin-Binding Photooxidative Stress-Inducible Aqueous Carotenoprotein from a Eukaryotic Microalga Isolated from Asphalt in Midsummer

Water-soluble orange carotenoid proteins (OCPs) that bind 3'-hydroxyechinenone are found in cyanobacteria, and are thought to play a key role in photoprotection. The distribution of OCPs in eukaryotes remains largely unknown. In this study, we identified a novel OCP that predominantly binds astaxanthin from a eukaryotic microalga, strain Ki-4, isolated from a dry surface of heated asphalt in midsummer. A purified astaxanthin-binding OCP, named AstaP, shows high solubility in water with an absorption peak at 484 nm, and possesses a heat-stable activity that quenches singlet oxygen. The deduced amino acid sequence of AstaP comprises an N-terminal hydrophobic signal peptide, fasciclin domains found in secreted and cell surface proteins, and N-linked glycosylation sites, the first example of a carotenoprotein among fasciclin family proteins. AstaP homologs of unknown function are distributed mainly in organisms from the hydrosphere, such as marine bacteria, cyanobacteria, sea anemone and eukaryotic microalgae; however, AstaP exhibits a unique extraordinarily high isoelectric point (pI) value among homologs. The gene encoding AstaP, as well as the AstaP peptide, is expressed abundantly under conditions of dehydration and salt stress in conjunction with high light exposure. As a unique aqueous carotenoprotein, AstaP will provide a novel function of OCPs in protection against extreme photooxidative stresses.

THU0058 B Cell Receptor Editing in Scleroderma Patients Generates Pathogenic Anti-PDGFR Autoantibodies

BackgroundSystemic sclerosis or scleroderma (SSc) is a clinically heterogeneous disease of the connective tissue characterized by vascular, immune/inflammatory and fibrotic manifestations. We have hypothesized that the serum of SSc patients...

Synthesis of mesoporous multiwall ZnO nanotubes by replicating silk and application for enzymatic biosensor

A facial biotemplate-directed synthetic route for fabricating mesoporous multiwall ZnO nanotubes (ZnO-MMNTs) was proposed. Owing to the mesoporous multiwall based matrix, one dimensional (1D) tubes based channels and high isoelectric point (IEP), the prepared ZnO-MMNTs are wonderful platform to immobilize glucose oxidase (GOx) for glucose biosensing. Scale-adaptive cells are constructed to hold enzymes molecules, maintain enzymatic activity and keep stability. The prepared enzymatic electrode (chitosan/GOx/ZnO/Au) exhibits high sensitivity (47.2μAmM−1cm−2), very fast response (<2s), quite low KMapp (1.09mM), excellent selectivity and stability. Therefore, ZnO-MMNTs are promising material for enzyme assembly and other biological applications.