Immobilized Enzyme Column Research Articles

Evaluation of Proteolytic Digestion Efficiency in Hydrogen Exchange-Mass Spectrometry Experiments Using the Digestible Peptide Score.

In a hydrogen exchange-mass spectrometry (HX-MS) experiment, the enzymatic proteolysis of the deuterated protein is an essential step. Often the differences in the performance between different digestion protocols or between immobilized protease columns can be challenging to evaluate. To compare differences in the performance of immobilized protease columns, a new digestion efficiency metric known as digestible peptide scoring (DPS) was developed and is presented in this work. The measured response fraction of substance P peptide is used to assign a value between 0% and 100% based on the fraction of substance P digested by the enzyme, using angiotensin II as an undigested internal standard. In this work, the DPS approach was tested using multiple immobilized pepsin batches prepared using different protocols. The results demonstrate the repeatability of DPS values for batches prepared using the same conditions and the ability of the DPS evaluations to provide unique values when the immobilization conditions were altered. Protein digestions obtained with a higher scoring column were better than digestions obtained using a lower scoring column. The DPS evaluation is simple and quickly provides an unambiguous assessment which can be used to evaluate an immobilized enzyme column's suitability prior to performing an experiment, to track performance over a column's lifetime, to optimize protease immobilization protocols specifically for the quench conditions of a particular experiment, and to optimize the digestion conditions.

Immobilized Enzyme Column Combined with HPLC and Column Switching Method for the Analysis of Complicated Matrix Such As Body Fluids

Copyright: © 2014 Shintani H. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Enzymes are bio-catalysts which affect transformation of substrates to products with high specificity. The usage of enzymes in domestic and industrial applications is well known and has been well documented since the early history of civilization. With the advances in understanding of enzymology, usage of enzymes in industrial and biotechnological processes and molecular medicine has proliferated.

Quantitation of the Cancer Marker LASA Using Immobilized Enzymes in a Semiautomated System

Cancer patients are found to have elevated serum levels of lipid-associated sialic acid (LASA). LASA measurement provides a valuable test for the diagnosis of human cancer. The currently used measurement methods are tedious and nonselective. Here we report a method based on immobilized enzyme minicolumns in a flow system for the analysis of this clinically important analyte. Three enzymes comprising two minicolumns are introduced online and the analyte in a precisely injected volume of 20 microL is made to pass through these immobilized enzyme columns. The final product of the reaction is detected amperometrically. Neuraminidase and NANA-aldolase are coimmobilized on controlled pore glass, followed by a column of pyruvate oxidase. The method was applied to the quantitation of LASA in the sera of normal individuals and patients with cancer of the esophagus. We observed no loss of activity of the immobilized enzymes after analyzing about 300 samples. The sample throughput was 30 samples per hour.

Quantitation of the cancer marker LASA using immobilized enzymes in a semiautomated system

Cancer patients are found to have elevated serum levels of lipid-associated sialic acid (LASA). LASA measurement provides a valuable test for the diagnosis of human cancer. The currently used measurement methods are tedious and nonselective. Here we report a method based on immobilized enzyme minicolumns in a flow system for the analysis of this clinically important analyte. Three enzymes comprising two minicolumns are introduced online and the analyte in a precisely injected volume of 20 microL is made to pass through these immobilized enzyme columns. The final product of the reaction is detected amperometrically. Neuraminidase and NANA-aldolase are coimmobilized on controlled pore glass, followed by a column of pyruvate oxidase. The method was applied to the quantitation of LASA in the sera of normal individuals and patients with cancer of the esophagus. We observed no loss of activity of the immobilized enzymes after analyzing about 300 samples. The sample throughput was 30 samples per hour.

Sensitive Determination of Reactive Oxygen Species by Chemiluminescence Methods and Their Application to Biological Samples and Health Foods

Sensitive and selective methods, based on chemiluminescence reactions, were introduced for determination of reactive oxygen species (ROS) and their applications to biological samples and health foods. First, a sensitive method for determination of H(2)O(2) by peroxyoxalate chemiluminescence (PO-CL) was developed. This method could be applied to determine small amounts of H(2)O(2) in cola drinks and bacterial contamination of food items. Secondly, the combination of immobilized enzyme column reactor, or ultraviolet irradiation system, with the PO-CL detection method was able to determine clinical substrates (i.e. choline-containing phospholipids, polyamines and D-amino acids) and organic peroxides. Also, an evaluation method of the quenching effect of luminol chemiluminescence against ROS was developed. The sensitive, rapid and precise measurement of the quenching effect against ROS such as superoxide, singlet oxygen, hydroxyl radical, peroxynitrite and hypochlorous ion was achieved. The proposed method could be applied to rosemary extracts, natural colorants and grape seed extracts.

High infusion pressure in conjunction with vitreous surgery alters the morphology and function of the retina of rabbits

To investigate the effects of high infusion pressure in conjunction with pars plana vitrectomy (PPV) on retinal morphology and function in rabbits. Pars plana vitrectomy was performed under urethane (0.8 mg/kg) anaesthesia in the right eye of albino rabbits following phacoemulsification and aspiration (PEA). The left eyes were not touched. After PEA, the animals were divided into two groups. In six eyes, intraocular pressure (IOP) was increased to 80 mmHg for 30 mins (high-pressure group) and in five eyes IOP was maintained at 40 mmHg for 30 mins (low-pressure group). The IOPs were regulated by the height of the bottle of balanced salt solution (BSS) and monitored with a pressure transducer. After the pressure elevation, vitreous fluid was collected to measure the glutamate concentration. Then, PPV was performed for 15 mins in both groups under an infusion pressure of 40 mmHg. In five additional rabbits, PEA alone was performed in the right eye, and vitreous fluid was collected (PEA group). Functional alterations were assessed by recording visual evoked potentials (VEPs) and electroretinograms (ERGs). Ten days after the IOP changes, the animals were killed with intravenous pentobarbital sodium and the eyes were prepared for histological analysis. Damage to retinal ganglion cells (RGCs) was quantified by counting the number of cells in the ganglion cell layer (GCL). The contralateral eyes in the high-pressure group served as controls (n = 6). The mean implicit time (IT) of the VEPs in the high-pressure group was significantly longer than that before the IOP elevation, by 114-124% (p < 0.05, paired t-test), and also than that of control eyes (p < 0.05, anova followed by t-test). No significant changes in the VEPs were detected in either the low-pressure group or the PEA group. There were significantly fewer cells in the GCL in the high-pressure group (24.7/mm) than in the control animals (41.4/mm; p < 0.05, Dunnett's test). The number of cells in the GCL in the low-pressure and PEA groups did not significantly differ to that in the controls. The amplitudes of the ERG a- and b-waves were not significantly changed (p > 0.05, paired t-test). These results suggest that high infusion pressure in conjunction with PPV leads to morphological and functional changes in the retina. The absence of ERG changes and presence of VEP changes suggest that these changes were due to damage to RGCs, which supports the morphological observations.

Fluorometric determination of urea in alcoholic beverages by using an acid urease column-FIA system

An acid urease column was applied to a fluorometric flow-injection analysis (FIA) system as a recognition element for determination of urea in rice wines. The acid urease has specific properties of showing its catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans. The enzymes were covalently immobilized onto porous glass beads with controlled pore size and then, packed into a small polymer column. The flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, and a flow-through quartz cell attached to a fluorescent spectrophotometer. Citrate buffer (50 mM, pH 5.0) as the carrier solution was continuously pumped through the system. Sample solutions were introduced into the system via a rotary injection valve. A standard urea solution was measured through monitoring variations in fluorescent intensity attributable to fluorescent isoindole derivatives formed by coupling with ammonia molecules released in the enzymatic hydrolysis of urea and orthophthalaldehyde reagents. The fluorescent intensity was measured under the conditions of λ ex = 415 nm and λ em = 485 nm. A wide, linear relationship was obtained between the concentration of urea (1.0–100 μM) and the variation in fluorescent intensity. The monitoring did not suffer from ethanol and various amino acids contained in rice wines. Real samples pretreated with ion exchange resins for removal of endogenous ammonia were introduced into the FIA system and urea in the samples was determined. These results were compared with those obtained with use of an F-kit method. The proposed FIA system should present sensitive, selective and convenient analysis of urea in alcoholic beverages.

An improved method for assaying phosphatidylcholine in mouse tissue

Introduction: To measure levels of phosphatidylcholine (PtdCh) in various mouse tissues, we developed a rapid and precise method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. To generate an example data set, the effect of methoxamine (an α 1-adrenergic agonist) on the PtdCh levels was examined by this method in the artery and the submandibular gland of the mouse in vivo. Methods: Under our modifications of the method of Zapata et al. [J. Neurosci. 18 (1998) 3597], the mixture of lipophilic choline metabolites (PtdCh, lyso-PtdCh, and sphingomyelin) extracted by chloroform from the tissue homogenate was dried without prior separation and hydrolyzed with free choline by a 1-N perchloric acid solution containing ethylhomocholine (an internal standard for choline assay) at 90 °C for 1 h. Subsequently, the hydrolyzed mixture was injected directly into the HPLC system for PtdCh assay. Results: The present method permitted PtdCh assay within 5 min in one chromatographic run. Recovery of an authentic PtdCh sample was 99% ( n=10). The within-run coefficients of variation for choline derived from PtdCh in the same tissue samples were 0.6% ( n=10) and 1.3% ( n=30). Under the present method, the lowest and highest PtdCh values in tissue samples were about 2 μmol/g (eye ball) and 29 μmol/g (spinal cord), respectively. Methoxamine significantly decreased PtdCh levels and increased free choline levels in mouse artery and submandibular gland. Discussion: Under the present sample processing procedure, the choline values originating from lyso-PtdCh and sphingomyelin were much less than those originating from PtdCh hydrolysis. Thus, it was possible to inject the hydrolyzed mixture directly into the HPLC system for PtdCh assay. Since the present method provides simple, rapid, and highly reliable PtdCh determination, it is suitable for routine assay of PtdCh in a large number of samples.

Spectrophotometric Microdetermination of Urea in a Rice Wine by Using an Immobilized Acid Urease Column FIA System

Determination of urea in rice wines was performed by using a spectrophotometric flow-injection analysis (FIA) system introducing an acid urease column as a recognition element. An acid urease, having specific properties of showing catalytic activity in low pH range and tolerance to ethanol in comparison to those of a urease from jack-beans, was covalently immobilized onto porous glass beads and then, packed into a small polymer column. This flow-type of the biosensing system was assembled with a sample injection valve, the immobilized enzyme column, a gas-diffusion unit, and a flow-through quartz cell attached to a UV/VIS detector. Standard urea solutions were measured through monitoring variations in absorbance resulting from pH shift due to ammonia molecules enzymatically generated. A wide, linear relationship was obtained between the concentration of urea (7.8 µM - 1.0 mM) and the change in absorbance. Followed by several investigations for application of this FIA system for measurement of urea in commercially available rice wines, the real samples were injected into the FIA system and urea in the samples were determined. These results were compared with those obtained with use of an F-kit method which was widely used for determination of urea. Comparative studies exhibited that this FIA system might be a powerful tool for urea determination in alcoholic beverages.

An improved method for assaying phosphocholine and glycerophosphocholine in mouse tissue

Introduction: To measure the levels of phosphocholine (PCh) and glycerophosphocholine (GPCh) in the tissues and organs of mice, we developed a simple and rapid method using high-performance liquid chromatography (HPLC) with electrochemical detection (ECD) and an immobilized enzyme column. Methods: Under our modifications of the separation procedure of Klein et al. [Neurochem. Int. 2 (1993) 293], PCh and GPCh in the hydrophilic phase of the homogenate samples were selectively hydrolyzed into free choline by alkaline phosphatase and a 0.4-N perchloric acid solution, respectively, and the resulting hydrolyzed mixtures were directly injected into the HPLC system for analysis. Results: The present method permits PCh or GPCh assay within 5 min in one chromatographic run. Recoveries from tissue samples were 97% for PCh and 101% for GPCh. The percentages of the crossover reaction to the authentic PCh and GPCh were 0.4% and 3.8%, respectively. The within-run coefficients of variation for choline derived from PCh and GPCh in the tissue samples were 1.2% and 1.4%, respectively. Discussion: The method is effective and has been applied to the measurement of PCh and GPCh levels in several tissues of mice.

Automated signature peptide approach for proteomics

This paper addresses the issue of automating the multidimensional chromatographic, signature peptide approach to proteomics. Peptides were automatically reduced and alkylated in the autosampler of the instrument. Trypsin digestion of all proteins in the sample was then executed on an immobilized enzyme column and the digest directly transferred to an affinity chromatography column. Although a wide variety of affinity columns may be used, the specific column used in this case was a Ga(III) loaded immobilized metal affinity chromatography (IMAC) column. Ga(III)–IMAC is known to select phosphorylated peptides. Phosphorylated peptides selected by the affinity column from tryptic digests of milk were automatically transferred to a reversed-phase liquid chromatography (RPLC) column. Further fractionation of tryptic peptides on the RPLC column was achieved with linear solvent gradient elution. Effluent from the RPLC column was electrosprayed into a time-of-flight mass spectrometer. The entire process was controlled by software in the liquid chromatograph. With slight modification, it is possible to add multiple columns in parallel at any of the single column positions to further increase throughput. Total analysis time in the tandem column mode of operation was under 2 h.

Flow injection amperometric enzyme biosensor for direct determination of organophosphate nerve agents.

A flow injection amperometric biosensor for the determination of organophosphate nerve agents was developed. The biosensor incorporated an immobilized enzyme reactor that contains the enzyme organophosphorus hydrolase covalently immobilized on activated aminopropyl controlled pore glass beads and an electrochemical flow-through detector containing carbon paste working electrode, a silver/silver chloride reference electrode, and stainless steel counter electrode. The organophosphorus hydrolase catalyzed the hydrolysis of organophosphate with nitrophenyl substituent to generate p-nitrophenol which is then detected downstream electrochemically at the carbon paste electrode poised at 0.9 V vs the reference electrode. The amperometric response of the biosensor was linear up to 120 microM and 140 microM, with lower detection limits of 20 nM and 20 nM, for paraoxon and methyl parathion, respectively. The response was very reproducible (RSD 2%, n = 35) and stable for over 1 month when the immobilized enzyme column was stored at 4 degrees C. Each assay took ca. 2 min giving a sample throughput of 30 h(-1). The applicability of the biosensor to monitor paraoxon and methyl parathion in distilled water and simulated well water was demonstrated.

Proteolysis of whole cell extracts with immobilized enzyme columns as part of multidimensional chromatography

This paper describes the efficacy of immobilized trypsin columns in the digestion of cellular extracts that contained thousands of proteins. Effectiveness of proteolysis was evaluated with extracts of Escherichia coli by size-exclusion chromatography. Immobilized trypsin columns were operated in either the continuous-flow or stopped-flow mode at temperatures ranging from ambient to 37°C with incubation times of 0–2 h. The results of these studies indicate that reduced and alkylated extracts of proteins from E. coli can be digested in 20 min when the enzyme column is operated at elevated temperature. The advantage of immobilized enzyme columns is that they can be easily incorporated into multidimensional separation systems for automated proteomics.

Automatic synthesis of l-[ β- 11C]amino acids using an immobilized enzyme column

We have developed a system for the automatic synthesis of l-[ β- 11C]amino acids for i.v. injection by means of enzyme-mediated reactions from 11CO 2 via 11CH 3I and d, l-[ β- 11C]alanine as labeled intermediates. This system, which incorporates an ultrafilter cartridge sterilized by electron beam irradiation and a column packed with immobilized enzymes, was effective for eliminating enzymes and endotoxins that may contaminate the product. Using this system, 1.3±0.5 GBq of 5-hydroxy- l-[ β- 11C]tryptophan with a radiochemical purity of 97.1±0.6% and a specific activity of 39.6±8 GBq/μmol a pH value of 4 could be obtained in about 32 min ( n=3, at EOS). No endotoxin, enzyme, or bacteria was detected in the product. l-[ β- 11C]dihydroxyphenylalanine ( l-[ β- 11C]DOPA) was also synthesized using this system.

Synthesis of L-[ β- 11C]amino acids using immobilized enzymes

L-[ β- 11C]-3,4-dihydroxyphenylalanine(L-[ β- 11C]DOPA) and L-[ β- 11C]-5-hydroxytryptophan(L-[ β- 11C]-5-HTP) were synthesized in one step with immobilized thermostable enzymes (alanine racemase, D-amino acid oxidase, and β-tyrosinase or tryptophanase) on an aminopropyl-CPG carrier in a single column and by passing D,L-[3- 11C]alanine through the column with coenzymes and other substrates. L-[ β- 11C]DOPA and L-[ β- 11C]-5-HTP could be obtained at yields of 53% and 60%, respectively, by optimizing the amounts and the ratios of the enzymes used, the reaction temperature, the pH, and the flow rate. Moreover, the same immobilized enzyme column could be used repeatedly.

Rapid verification of disulfide linkages in recombinant human growth hormone by tandem column tryptic mapping

An automated tryptic mapping method was developed for characterization of disulfide linkages in recombinant human growth hormone (rhGH). The hormone was trypsin digested and the peptide fragments concentrated by eluting rhGH through an immobilized trypsin column and transferring the peptides directly to a reversed-phase liquid chromatography (RP-LC) column where they were collected. Reaction time was controlled by the flow-rate. Following tryptic digestion of a sample, the immobilized enzyme column was uncoupled from the flow train by a switching valve and the RP-LC column eluted with a solvent gradient ranging from 0.1% trifluoroacetic acid (TFA) with 1% acetonitrile (ACN) to ACN with 0.1% TFA and 5% water. This two-step mapping process was achieved within 2 h on both native and reduced rhGH samples. The chromatographic elution position and mass spectra matrix-assisted laser desorption ionization time-of-flight mass spectrometry of native rhGH and sulfur-containing peptides were determined with standards. Standards of the individual sulfhydryl (–SH) containing peptides and all possible disulfide linked peptides that could result from coupling the –SH peptides in disulfide linkages were obtained by synthesis and chromatographic purification. This approach allowed the chromatographic elution position of all possible mismatched disulfide containing peptides to be established and samples of rhGH to be examined for improper folding.

A Sensitive and Rapid FIA with an Immobilized Enzyme Column Reactor and Peroxyoxalate Chemiluminescence Detection for the Determination of Total d-Amino Acids in Human Plasma

A sensitive and rapid FIA using an immobilized enzyme column reactor was developed for the determination of D-amino acids in human plasma. This method is based on a specific enzymic reaction with D-amino acid oxidase and a sensitive peroxyoxalate chemiluminescence (PO-CL) detection with bis(2, 4, 6-trichlorophenyl)oxalate and 2, 4, 6, 8- tetrathiomorpholinopyrimido[5, 4-d]pyrimidine as CL reagents. Eleven kinds of D-amino acids could be independently detected quantitatively (r=0.983-1.000). The detection limits of each D-amino acid ranged from 0.4 to 30pmol/10μl injection at a signal-to-noise ratio of 3. The average concentration level of D-amino acids in human plasma was found to be 14.6±4.7 (in the range of 5.0-24.3, n=18)nmol/ml plasma. The correlation between the results obtained by the proposed FIA-PO-CL method and the colorimetric method was satisfactory (r=0.900). Moreover, this method is advantageous, since the net volume of the plasma sample needed was 2μl for each run; also, 5 samples could be analyzed within 10min.

Determination of Damaged Starch and Diastatic Activity in Wheat Flour Using a Flow‐Injection Analysis Biosensor Method

ABSTRACTThe determination of damaged starch and diastatic activity in flour was studied using a flow‐injection analysis (FIA) biosensor system. The system consisted of an oxygen electrode and an immobilized enzyme column containing purified glucoamylase and glucose oxidase immobilized on activated aminopropyl glass beads. The biosensor system has an optimum pH between 6.5 and 7.5 and an optimum temperature of 35°C for glucose measurement. The response of the FIA biosensor was linear up to 1.000 g/L of glucose with a lower detection limit of 0.025 g/L. Each assay took about 20 min, and the system showed good reproducibility (r = 0.998, n = 8). When applied to the measurement of damaged starch and diastatic activity in wheat flour, the results obtained agreed with those obtained using the conventional methods of measurement. This biosensor system is a rapid practical alternative for the measurement of damaged starch and diastatic activity in wheat flour.

Flow-injection chemiluminescent determination of glycerol-3-phosphate and glycerophosphorylcholine using immobilized enzymes.

Flow injection procedures with immobilized enzyme mini-columns are described for the determination of glycerol-3-phosphate, and glycerophosphorylcholine with chemiluminescent detection. The hydrogen peroxide produced on-line is coupled with a luminol (5-amino-2,3,-dihydro-1,4-phthalazinedione) peroxidation chemiluminescent system. The detection limits for glycerol-3-phosphate and glycerophosphorylcholine are 5 x 10(-7) M and 1 x 10(-6) M respectively with r.s.d. < 2%. The sample throughput is 40/h. The immobilized enzyme columns did not show any deterioration in activity after usage for 3 months.

Kinetics of glucose isomerization to fructose by immobilized glucose isomerase in the presence of substrate protection

The activity of immobilized glucose isomerase of Streptomyces murinus has been tested batchwise under different conditions in order to gather the related kinetic parameters necessary to optimize an immobilized enzyme column for the continuous production of high fructose corn syrup (HFCS). To this purpose, the Briggs-Haldane model incorporating an apparent first-order inactivation constant has been used with success. A comparison of the equilibrium constants and of the maximum theoretical conversion yields calculated at different temperatures with those estimated for the native enzyme demonstrates that the immobilization favours the transformation of glucose to fructose only at T > 70 °C, as a possible consequence of a combined effect of catalysis and equilibrium thermodynamics enhancement. Enzyme inactivation has also been tested at different temperatures and sugar concentrations to evaluate the related kinetic parameters under different conditions of substrate protection.