High-performance affinity chromatography of NADP+ dehydrogenases from cell-free extracts using a nucleotide analogue as general ligand.

An epoxy-activated silica column (50 cm x 0.45 cm I.D.) was derivatized with 8-[6-aminohexyl)amino]-2'-phosphoadenosine-5'-diphosphoribose; the bound ligand concentration was 11.4 mumol/g of dry silica, and the useful loading capacity was 2.3 mg of glutathione reductase. The new high-performance liquid chromatographic column specifically retained NADP(+)-dependent enzymes, which were quantitatively eluted specifically by NADP+ or, with better resolution, by potassium chloride. The new high-performance liquid chromatographic support was applied to the purification of glutathione reductase and glucose-6-phosphate dehydrogenase from cell-free extracts of baker's yeast, fish liver and rabbit hemolysates, with high recoveries and excellent purification factors.     

Kinetics of befunolol reductase from rabbit liver

The kinetic mechanism for the reduction of befunolol catalyzed by befunolol reductase from rabbit liver was investigated. From the initial velocity analysis, product inhibition and coenzyme binding studies, the reduction of befunolol was found to proceed through an ordered Bi Bi mechanism, in which beta-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH) binds to the enzyme firstly and NADP+ leaves lastly. NADPH bound to the free enzyme at a molar ratio of 1:1. Furthermore, the result of dead-end inhibition by Cibacron blue F3GA, a nucleotide analogue which binds to many enzymes, was consistent with the ordered Bi Bi mechanism for the enzyme.     

Electrolytic process for regenerating nadp from nadph in a polymer matrix-bound form

Regeneration of nicotinamide adenine dinucleotide phosphate (NADP) from its reduced form (NADPH) was performed in a matrix-bound form by an electrolytic method. NADP was immobilized to alginic acid. No significant loss of coenzymic function was induced by the immobilization of NADP on the matrix. Bound NADP was soluble in water. Glucose-6-phosphate dehydrogenase (G-6-PDH) was taken as a model system of coenzyme requiring enzyme. G-6-PDH immobilized on alumina particles was coupled with the soluble form of bound NADP in a fluidized bed type of reactor. The enzymatically reduced coenzyme was electrolytically oxidized in the coenzyme regenerator of NADP from NADPH, which was found to cause no harmful loss of coenzymic function.     

Evaluation of a monolithic epoxy silica support for penicillin G acylase immobilization

In this work, a new type of penicillin G acylase (PGA)-based monolithic silica support was developed and evaluated for the chiral separation in HPLC. The preparation procedure consisted of two steps: preparation of an epoxy derivatized monolithic silica column and chemical modification of the epoxide groups with the enzyme chiral selector. The epoxy Silica-Rod column for the immobilization of PGA was prepared with the in situ modification process by using epoxy-silanes and the identification of the species bound to the surface was achieved by solid-state nuclear magnetic resonance. The enzyme was covalently immobilized to the surface of the derivatized monolithic column. The enantioselectivity and the performance of the developed column are discussed and compared to the corresponding experimental data obtained with a PGA-based microparticulate (5 microm) silica column.     

Purification of NAD glycohydrolase from Neurospora crassa conidia by a polyclonal immunoadsorbent.

NAD glycohydrolase from Neurospora crassa conidia was purified by affinity chromatography on a column of polyclonal antibodies bound to an agarose matrix. The procedure was easy, non-denaturating and suitable for repetitive use of the gel. The enzyme obtained appeared homogeneous by sodiumdodecyl sulphate-polyacrylamide gel electrophoresis.     

Molecular recognition between protein and nicotinamide dinucleotide in intact, proton-translocating transhydrogenase studied by ATR-FTIR Spectroscopy

Nicotinamide dinucleotide binding to transhydrogenase purified from Escherichia coli was investigated by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy. Detergent-free transhydrogenase was deposited as a thin film on an ATR prism, and spectra were recorded during perfusion with buffers in the presence and absence of dinucleotide (NADP(+), NADPH, NAD(+), or NADH) in both H(2)O and D(2)O media. IR spectral changes were attributable to the bound dinucleotides and to changes in the protein itself. The dissociation constant of NADPH was estimated to be approximately 5 muM from a titration of the magnitude of the IR changes against the nucleotide concentration. IR spectra of related model compounds were used to assign principle bands of the dinucleotides. This information was combined with IR data on amino acids and with protein crystallographic data to identify interactions between specific parts of the dinucleotides and their binding sites in the protein. Several IR bands of bound nucleotide were sharpened and/or shifted relative to those in aqueous solution, reflecting a restriction to motion and a change in environment upon binding. Alterations in the protein secondary structure indicated by amide I/II changes were distinctly different for NADP(H) and for NAD(H) binding. The data suggest that NADP(H) binding leads to perturbation of a deeply buried part of the polypeptide backbone and to protonation of a carboxylic acid residue.     

Enzymatic synthesis of caged NADP cofactors: aqueous NADP photorelease and optical properties

The synthesis of caged NADP analogues 18, 19, and 20 has been accomplished by utilizing the transglycosidase activity of solubilized NAD glycohydrolase (porcine brain) to incorporate caged nicotinamides 2, 3, and 4 into NADP. The synthesis of several nicotinamides modified at the carboxamide with o-nitrobenzyl photolabile groups is demonstrated as well as their potential for enzymatic transglycosidation. These results further demonstrate the feasibility of direct enzymatic transglycosidation of sterically hindered substrates into NAD(P), although high nicotinamide analogue water solubility was found to be a necessary trait for yield enhancement with certain analogues. Caged analogues were surveyed under aqueous conditions for net NADP photorelease, while the UV and fluorescent properties of both analogues and their photobyproducts were assessed for compatibility with systems that rely on optical monitoring of enzyme activity. A highly water-soluble alpha-methyl-o-nitrobenzyl group 8 was developed for the synthesis of 20 in order to enhance net NADP photorelease. Compound 20 demonstrated a high 75% net NADP photoreleased without substantial UV optical blackening or fluorescent byproducts. Analogues 18 and 19 were shown by ESI/MALDI-MS to photogenerate primarily adducts of NADP with deleterious UV and fluorescent properties. Our work stresses the superior release properties conferred by alpha-methyl substitution on aqueous carboxamide photorelease from o-nitrobenzyl compounds.     

Sensitivity of steroid enzyme immunoassays. Comparison of four label enzymes in an assay system using a monoclonal anti-steroid antibody

The sensitivities of monoclonal antibody-based enzyme immunoassays for 11-deoxycortisol using alkaline phosphatase (AP), horseradish peroxidase (HRP), beta-galactosidase (beta-GAL) and glucose oxidase (GOD) as labels were compared. The anti-11-deoxycortisol antibody used was that produced in ascites by inoculating antibody-secreting hybridoma cells into mice. Enzyme labeling of 11-deoxycortisol was carried out by the N-succinimidyl ester method. The activated ester of 4-(2-carboxyethylthio)-11-deoxycortisol was treated with each enzyme to give a homologous enzyme-labeled antigen. In the competitive immunoassay, the bound and free enzyme-labeled antigens were separated by a double antibody method and the enzymic activity of the immune precipitate was determined by colorimetric and fluorimetric methods. The AP activity was measured in three ways, using p-nitrophenyl phosphate, nicotinamide adenine dinucleotide phosphate (NADP), and 4-methylumbelliferyl phosphate as substrates. o-Nitrophenyl beta-D-galactopyranoside and 4-methylumbelliferyl beta-D-galactopyranoside were used for beta-GAL, and 3,3',5,5'-tetramethylbenzidine (TMB) and 3-(p-hydroxyphenyl)propionic acid (HPPA) for HRP. In the case of GOD, TMB and HPPA were used in combination with HRP. A dose-response curve with a high sensitivity was obtained in each 11-deoxycortisol assay system by the use of a minimum amount of the enzyme-labeled antigen at an appropriate dilution of monoclonal anti-11-deoxycortisol antibody (Ka = 2 x 10(10) M-1). The amounts of 11-deoxycortisol needed to displace 50% of the bound label ranged from 5 to 15 pg in the colorimetric methods, and 4-9 pg in the fluorimetric methods.(ABSTRACT TRUNCATED AT 250 WORDS)     

Affinity Chromatography of Mushroom Tyrosinase

Abstract

The purification by column chromatography of a phenol-oxidizing enzyme, mushroom tyrosinase, was investigated using solid phase adsorbents designed to have specific affinity for the enzyme. Sepharose 4B, aminophenyl-bearing porous glass, and p-aminobenzylcellulose were chemically modified to introduce phenolic, catecholic, or benzoic groups on the polymer surface. The resulting preparations were tested for their effectiveness in separating tyrosinase from an impure protein mixture. The phenolic and benzoic polymers displayed no specific affinity for tyrosinase. Aminophenyl glass, with or without an attached phenolic group, adsorbed appreciable quantities of protein nonspecif-ically, thus complicating studies of its tyrosinase affinity properties. Dopamine, a dihydroxyphenyl derivative, was bound to Sepharose and was found to be effective in retaining tyrosinase at pH 5.5; elution of the enzyme by washing at pH 8.8 resulted in its purification by a factor of 10 to 14. Enzymatic oxidation of the adsorbent limited the number of purification cycles which could be carried out on a single column.     

Protein-induced excited-state dynamics of protochlorophyllide

The light-driven NADPH:protochlorophyllide oxidoreductase (POR) is a key enzyme of chlorophyll biosynthesis in angiosperms. POR's unique requirement for light to become catalytically active makes the enzyme an attractive model to study the dynamics of enzymatic reactions in real time. Here, we use picosecond time-resolved fluorescence and femtosecond pump-probe spectroscopy to examine the influence of the protein environment on the excited-state dynamics of the substrate, protochlorophyllide (PChlide), in the enzyme/substrate (PChlide/POR) and pseudoternary complex including the nucleotide cofactor NADP(+) (PChlide/NADP(+)/ POR). In comparison with the excited-state processes of unbound PChlide, the lifetime of the thermally equilibrated S(1) excited state is lengthened from 3.4 to 4.4 and 5.4 ns in the PChlide/POR and PChlide/NADP(+)/POR complex, whereas the nonradiative rates are decreased by ～30 and 40%, respectively. This effect is most likely due to the reduced probability of nonradiative decay into the triplet excited state, thus keeping the risk of photosensitized side reactions in the enzyme low. Further, the initial reaction path involves the formation of an intramolecular charge-transfer state (S(ICT)) as an intermediate product. From a strong blue shift in the excited-state absorption, it is concluded that the S(ICT) state is stabilized by local interactions with specific protein sites in the catalytic pocket. The possible relevance of this result for the catalytic reaction in the enzyme POR is discussed.     

Integrated, electrically contacted NAD(P)+-dependent enzyme-carbon nanotube electrodes for biosensors and biofuel cell applications

Integrated, electrically contacted beta-nicotinamide adenine dinucleotide- (NAD(+)) or beta-nicotinamide adenine dinucleotide phosphate- (NADP(+)) dependent enzyme electrodes were prepared on single-walled carbon nanotube (SWCNT) supports. The SWCNTs were functionalized with Nile Blue (1), and the cofactors NADP(+) and NAD(+) were linked to 1 through a phenyl boronic acid ligand. The affinity complexes of glucose dehydrogenase (GDH) with the NADP(+) cofactor or alcohol dehydrogenase (AlcDH) with the NAD(+) cofactor were crosslinked with glutaric dialdehyde and the biomolecule-functionalized SWCNT materials were deposited on glassy carbon electrodes. The integrated enzyme electrodes revealed bioelectrocatalytic activities, and they acted as amperometric electrodes for the analysis of glucose or ethanol. The bioelectrocatalytic response of the systems originated from the biocatalyzed oxidation of the respective substrates by the enzyme with the concomitant generation of NAD(P)H cofactors. The electrocatalytically mediated oxidation of NAD(P)H by 1 led to amperometric responses in the system. Similarly, an electrically contacted bilirubin oxidase (BOD)-SWCNT electrode was prepared by the deposition of BOD onto the SWCNTs and the subsequent crosslinking of the BOD units using glutaric dialdehyde. The BOD-SWCNT electrode revealed bioelectrocatalytic functions for the reduction of O(2) to H(2)O. The different electrically contacted SWCNT-based enzyme electrodes were used to construct biofuel cell elements. The electrically contacted GDH-SWCNT electrode was used as the anode for the oxidation of the glucose fuel in conjunction with the BOD-SWCNT electrode in the presence of O(2), which acted as an oxidizer in the system. The power output of the cell was 23 muW cm(-2). Similarly, the AlcDH-SWCNT electrode was used as the anode for the oxidation of ethanol, which was acting as the fuel, with the BOD-SWCNT electrode as the cathode for the reduction of O(2). The power output of the system was 48 microW cm(-2).     

Purification of hydrophilic and hydrophobic peptide fragments on a single reversed phase high performance liquid chromatographic column

Hydrophilic peptides generated from enzymic fragmentation of proteins are difficult to purify because they are either weakly bound or unretained by the reversed phase C₁₈ columns favoured for liquid chromatographic separation of peptide mixtures. To overcome this difficulty, peptides that were not bound or only weakly bound by a C₁₈ RP column were reacted with phenyl isothiocyanate (PITC), as used in the initial step in Edman sequencing. The hydrophobic phenylthiocarbamyl (PTC) peptide derivatives produced by the reaction were rechromatographed on the same column. Peptides generated by tryptic digestion of equine cytochrome C were used as a model system to test whether a complete set of peptide fragments could be purified by this method using just one column and solvent system. All the expected hydrophobic tryptic peptides bound to the RP column and were resolved by elution with acetonitrile, but no hydrophilic peptides were recovered as pure fractions. The column breakthrough fraction was reacted with PITC and rechromatographed on the same column, producing a profile consisting of 19 bound peaks. Further rechromatography of some of the fractions at different column temperatures enabled all six of the expected hydrophilic peptides to be purified and identified. The technique has also been applied to the sequence determination of coat protein from peanut stripe potyvirus protein, eight hydrophilic tryptic peptides being recovered and identified as PTC derivatives.     

Highly selective and sensitive detection of NADP coenzymes using co-immobilized glucose-6-phosphate dehydrogenase/diaphorase reactors as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system

Two enzyme reactors prepared by the co-immobilization of two different glucose-6-phosphate dehydrogenases (G6PDH; from Leuconstoc mescenteroides (LM) and yeast (Y) and diaphorase are employed to enhance the sensitivity of NAD(P) coenzymes as on-line amplifiers based on substrate recycling in a chemiluminometric flow-injection system. The NAD(P) coenzymes are recycled enzymatically during passage through the reactor in the presence of sufficient glucose-6-phosphate and oxygen in the carrier solution to produce a large amount of hydrogen peroxide, which is detected chemiluminometrically in the subsequent flow line. The G6PDH(LM)/diaphorase co-immobilized reactor is not specific between the NAD and NADP coenzymes, but shows a six fold selectivity towards NADP coenzymes compared to NAD coenzymes; the amplification factors for NAD and NADP coenzymes are 60 and 380, respectively, at a flow rate of 0.3 ml min(-1). In contrast, the G6PDH(Y)/diaphorase co-immobilized reactor is specific for NADP coenzymes with an amplification factor of about 600 (at a flow rate of 0.3 ml min(-1)). The detection limit is 6 fmol for both NADP(+) and NADPH.     

Immobilization of Amylases on Silica Support to Study Breakdown Products of Potato Starch by HPLC

Abstract

Immobilization of α- and β-amylases on epoxypropylsilanized PartiSphere-5 was achieved. Hydrolysis of 2% potato starch solution yielded limit dextrin on α-amylase bound column while a mixture of limit dextrin, maltose and glucose was obtained from β-amylase bound column. The β-amylase bound column converted limit dextrin from α-amylase column into glucose.     

High-performance catalytic chromatography. The adapter approach

A sequence-specific DNA that binds EcoRI endonuclease was immobilized on glycidioloxypropyl-silica and Sepharose by cyanogen bromide (CNBr)-activated coupling. Elution of bound enzyme by conventional affinity strategies (increase of salt concentration) or by catalysis-induced elution (adding a Mg2+ cofactor required for catalysis) was compared. Greater yield and fold-purification was obtained with catalysis-induced elution for both DNA-silica and DNA-Sepharose columns, and silica gives higher performance than Sepharose. Sodium dodecylsulfate polyacrylamide gel electrophoresis showed primarily a single band for EcoRI endonuclease for catalysis-induced elution from DNA-silica columns. Since catalysis-induced elution decreases the lifetime of DNA affinity columns, an alternative approach for preparing re-usable DNA columns was also developed. In this approach, a single stranded adapter DNA sequence is first coupled to silica or Sepharose and then annealed with another DNA sequence that contains a complementary, single stranded tail and the duplex binding site for EcoRI endonuclease. After use, replacing the hydrolyzed DNA regenerates the column. For this adapter approach, Sepharose gives better purity than silica and comparable yields and catalytic based elution gave the highest purity and yield, regardless of support. Substrate DNA with either a tail (for annealing to the column) at one end or both ends were compared and the former gave higher purity. Finally, enzyme binding to the substrate in solution ("trapping") or on a pre-bound substrate column was compared and trapping gave higher yield and similar purity to the alternative. Thus, trapping with a single tailed substrate oligonucleotide on a Sepharose adapter column and using catalytic elution gave the highest performance.     

Principles of multienzyme purifications by affinity chromatography

Recently in our laboratory, up to 20 different enzymes and their genetic variants have been purified from mouse andDrosophila by affinity chromatography. By virtue of the specific coenzyme requirements, up to ten different enzymes could be copurified from a single tissue extract either by biospecific elutions with different coenzymes or inhibitors, or by sequential passages of the extract through several cofactor-related affinity columns. Important principles were developed to purify enzymes exhibiting low affinity to the affinity columns. By “affinity filtration” of the extract through the affinity column, enzymes of low affinity can be retarded and separated effectively from strongly bound and nonadsorbed proteins. By the “saturation readsorption” procedure, enzymes of low affinity could be effectively separated from those of high affinity by overloading of the extracts on the affinity columns. Readsorption of the leaked low affinity enzymes to a second affinity column often results in better enzyme purification because of the elimination of competitive high affinity enzymes. With the application of these principles, the following enzymes and their genetic variants were highly purified via a single- or two-step affinity column procedure: lactate dehydrogenase-A, lactate dehydrogenase-B, lactate dehydrogenase-X, phosphoglycerate kinase-A, phosphoglycerate kinase-B, cytoplasmic and mitochondrial isocitrate dehydrogenase, malate dehydrogenase, malic enzyme, glucose-6-phosphate dehydrogenase, glutathione reductase, phosphoglucose isomerase and pyruvate kinase from mouse tissues; alcohol dehydrogenase, malate dehydrogenase, α-glycerol-phosphate dehydrogenase, malic enzyme, and glucose-6-phosphate dehydrogenase fromDrosophila.     

Robotic enzyme amplification: a comparison of some kinetic properties of bovine liver, Candida utilis and Proteus sp. glutamic dehydrogenases.

NADP(H)-specific Bakers yeast glucose 6-phosphate dehydrogenase (BYG6PDH) was paired, in turn, with each of three different source glutamate dehydrogenases (GDHs): NAD(P)-specific bovine liver (BLGDH), NADP-specific Candida utilis (CUGDH) and NADP-specific Proteus sp. (PSGDH) to constitute three enzyme cycling systems; (i) BYG6PDH/BLGDH; (ii) BYG6PDH/CUGDH; and (iii) BYG6PDH/PSGDH. When incorporated into an enzymatic cycling/amplification system for NAD kinase and run on a centrifugal fast analyzer (CFA), the microbial source enzyme CUGDH gave rise to a seven-fold greater amplification rate [21.5 x 10(3) cycles-1 (cph)] relative to that realized (3 x 10(3) cph) using the BYG6PDH/BLGDH cycling pair previously reported. Either of these cycling systems can be used as a flexible and general-purpose module for robotic amplification and data collection of NADP(H) linked enzymes as a user's requirements dictate. Although the BYG6PDH/PSGDH cycling pair produced a respectable cycling rate (14.4 x 10(3) cph), for reasons discussed the PSGDH enzyme was not considered a suitable replacement for BLGDH in an NADP(H) cycling system.     

Interaction of homogeneous mitochondrial ATPase from rat liver with adenine nucleotides and inorganic phosphate.

Mitochondrial ATPase from rat liver mitochondria contains multiple nucleotide binding sites. At low concentrations ADP binds with high affinity (1 mole/mole ATPase, KD = 1-2 muM). At high concentrations, ADP inhibits ATP hydrolysis presumably by competing with ATP for the active site (KI = 240-300 muM). As isolated, mitochondrial ATPase contains between 0.6 and 2.5 moles ATP/mole ATPase. This "tightly bound" ATP can be removed by repeated precipitations with ammonium sulfate without altering hydrolytic activity of the enzyme. However, the ATP-depleted enzyme must be redissolved in high concentrations of phosphate to retain activity. AMP-PNP (adenylyl imidodiphosphate) replaces tightly bound ATP removed from the enzyme and inhibits ATP hydrolysis. AMP-PNP has little effect on high affinity binding of ADP. Kinetics studies of ATP hydrolysis reveal hyperbolic velocity vs. ATP plots, provided assays are done in bicarbonate buffer or buffers containing high concentrations of phosphate. Taken together, these studies indicate that sites on the enzyme not directly associated with ATP hydrolysis bind ATP or ADP, and that in the absence of bound nucleotide, Pi can maintain the active form of the enzyme.     

Mode change in the self-motion of a benzoquinone disk coupled with a NADPH system

The self-motion of a benzoquinone (BQ) disk on NADPH was investigated as the coupling of an autonomous motor and an enzyme reaction. In the absence of the enzyme reaction, features of motion changed depending on the concentration of NADPH, that is, continuous motion→ intermittent oscillatory motion→ no motion. When the reverse reaction from NADP(+) to NADPH was introduced into the system with the addition of an enzyme reaction, continuous motion changed to intermittent oscillatory motion with small amplitude. The mechanism of this mode change is discussed in relation to the surface tension as a driving force and the time course of UV spectra as a window to the progress of the reaction. Characteristic features of the mode change were qualitatively reproduced by a numerical calculation.     

Production of monoclonal antibodies against a cell surface concanavalin A binding glycoprotein

Concanavalin A-binding (Con-A)-binding cell surface glycoproteins were isolated, via Con A-affinity chromatography, from Triton X-100-solubilized Chinese hamster ovary (CHO) cell plasma membranes. The Con A binding glycoproteins isolated in this manner displayed a significantly different profile on sodium dodecyl sulfate--polyacrylamide gels than did the Triton-soluble surface components, which were not retarded by the Con-A-Sepharose column. [125I]-Con A overlays of the pooled column fractions displayed on sodium dodecyl sulfate--polyacrylamide gel electro-phoresis (SDS-PAGE) demonstrated that there were virtually no Con A receptors associated with the unretarded peak released by the Con A-Sepharose column, whereas the material which was bound and specifically eluted from the Con A-Sepharose column with the sugar hapten alpha-methyl-D-mannopyranoside contained at least 15 prominent bands which bound [125I]-Con A. In order to produce monoclonal antibodies against various cell surface Con A receptors, Balb/c mice were immunized with the pooled Con A receptor fraction. Following immunization spleens were excised from the animals and single spleen cell suspensions were fused with mouse myeloma P3/X63-Ag8 cells. Numerous hybridoma clones were subsequently picked on the basis of their ability to secrete antibody which could bind to both live and glutaraldehyde-fixed CHO cells as well as to the Triton-soluble fraction isolated from the CHO plasma membrane fraction. Antibody from two of these clones was able to precipitate a single [125I]-labeled CHO surface component of approximately 265,000 daltons.