Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Forde, Jessica; Oakey, Laura; Jennings, Linda W.; Mulcahy, Patricia

doi:10.1016/j.ab.2004.11.016

Cited by 9 publications

(12 citation statements)

References 19 publications

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“…Nucleotide cofactors like NAD + , NADPH, and ATP are used to selectively elute bound dehydrogenases and kinases from the matrix [ 39 ]. The "kinetic locking-on" is an important strategy in selective enzyme binding and elution-it has been successfully used to purify some dehydrogenases [ 10 ]. If and when a dye-ligand mimics a substrate or a cofactor (like NAD + or NADP + ), then the enzyme could be locked on to the dye adsorbent, by including the other substrate in the loading and equilibration buffers.…”

Section: Binding and Elution Strategiesmentioning

confidence: 99%

High-Throughput Screening of Dye-Ligands for Chromatography

Kumar¹,

Punekar²

2014

Methods in Molecular Biology

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Dye-ligand-based chromatography has become popular after Cibacron Blue, the first reactive textile dye, found application for protein purification. Many other textile dyes have since been successfully used to purify a number of proteins and enzymes. While the exact nature of their interaction with target proteins is often unclear, dye-ligands are thought to mimic the structural features of their corresponding substrates, cofactors, etc. The dye-ligand affinity matrices are therefore considered pseudo-affinity matrices. In addition, dye-ligands may simply bind with proteins due to electrostatic, hydrophobic, and hydrogen-bonding interactions. Because of their low cost, ready availability, and structural stability, dye-ligand affinity matrices have gained much popularity. Choice of a large number of dye structures offers a range of matrices to be prepared and tested. When presented in the high-throughput screening mode, these dye-ligand matrices provide a formidable tool for protein purification. One could pick from the list of dye-ligands already available or build a systematic library of such structures for use. A high-throughput screen may be set up to choose best dye-ligand matrix as well as ideal conditions for binding and elution, for a given protein. The mode of operation could be either manual or automated. The technology is available to test the performance of dye-ligand matrices in small volumes in an automated liquid-handling workstation. Screening a systematic library of dye-ligand structures can help establish a structure-activity relationship. While the origins of dye-ligand chromatography lay in exploiting pseudo-affinity, it is now possible to design very specific biomimetic dye structures. High-throughput screening will be of value in this endeavor as well.

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Section: Binding and Elution Strategiesmentioning

confidence: 99%

High-Throughput Screening of Dye-Ligands for Chromatography

Kumar¹,

Punekar²

2014

Methods in Molecular Biology

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“…Similar results were obtained with NAD + derivative III and NAD + derivative I, with the former having a max at 260 nm with a second peak at 225 nm. A comparison of spectral scans of NAD(P) + derivatives I-IV with N 6 -linked immobilized NAD(P) + derivatives synthesized using N 6 -carboxymethyl NAD(P) + [3] is presented in Fig. 1.…”

Section: Immobilized Nad(p) + Derivatives Synthesized By Carbodiimidementioning

confidence: 99%

“…It is also important to note that although Sporosarcina sp. LPheDH does not lock on to either S 6 -or 8Ј-azo-linked NAD + , D-phenylalanine has been used successfully as a soluble locking-on/KBEC ligand for L-PheDH provided that N 6 -linked NAD + is used [3]. Relevant kinetic Table 3 Relevant kinetic constants for soluble KBEC ligands of YADH, L-LDH, GDH, and L-PheDH…”

Section: Evaluation Of Nad(p) + Derivatives I-iv For Use With Kbec Stmentioning

confidence: 99%

“…The assay conditions for GDH were 50 mM potassium phosphate, pH 7.4, 30°C, 1.0 mM NAD + . b Taken from [3]. The assay conditions for L-PheDH were 100 mM glycine containing 0. constants for the KBEC ligands are presented in Table 3.…”

Section: Evaluation Of Nad(p) + Derivatives I-iv For Use With Kbec Stmentioning

confidence: 99%

“…G-6-P-DH shows a strong aYnity for N 6 -linked immobilized NADP + and binds to the aYnity matrix even in the absence of KBEC ligands [3,9]. GDH from both bovine liver and C. utilis can be locked on to N 6 -linked immobilized NADP + derivatives when glutarate is used as the soluble KBEC ligand [9].…”

Section: Evaluation Of Nad(p) + Derivatives I-iv For Use With Kbec Stmentioning

confidence: 99%

See 2 more Smart Citations

Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+

Oakey

Mulcahy

2004

Analytical Biochemistry

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Amperometric Ethanol Biosensors Based on Chitosan‐NAD⁺‐Alcohol Dehydrogenase Films

Zhang¹,

Gorski²

2011

Electroanalysis

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The reagentless and oxygen-independent biosensors for ethanol were developed based on the covalent immobilization of alcohol dehydrogenase (ADH) and its cofactor nicotinamide adenine dinucleotide (NAD + ) on chitosan (CHIT) chains. The CHIT-NAD + -ADH structures were adsorbed onto carbon nanotubes (CNT) in order to provide a signal transduction based on the recycling of redox states of NAD cofactor at CNT (detection limit, 8-30 mM ethanol; dynamic range up to 20 mM). The CHIT-NAD + -dehydrogenase/CNT hybrid material represents a general approach to the development of dehydrogenases-based electrochemical biosensors. Interestingly, the CHIT-NAD + solutions preserved their enzymatic activity even after five years of storage at 4 8C.

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Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Cited by 9 publications

References 19 publications

High-Throughput Screening of Dye-Ligands for Chromatography

High-Throughput Screening of Dye-Ligands for Chromatography

Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+

Amperometric Ethanol Biosensors Based on Chitosan‐NAD⁺‐Alcohol Dehydrogenase Films

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Fundamental differences in bioaffinity of amino acid dehydrogenases for N6- and S6-linked immobilized cofactors using kinetic-based enzyme-capture strategies

Cited by 9 publications

References 19 publications

High-Throughput Screening of Dye-Ligands for Chromatography

High-Throughput Screening of Dye-Ligands for Chromatography

Immobilized cofactor derivatives for kinetic-based enzyme capture strategies: direct coupling of NAD(P)+

Amperometric Ethanol Biosensors Based on Chitosan‐NAD+‐Alcohol Dehydrogenase Films

Contact Info

Product

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Amperometric Ethanol Biosensors Based on Chitosan‐NAD⁺‐Alcohol Dehydrogenase Films