Spacer arms in affinity chromatography: Use of hydrophilic arms to control or eliminate nonbiospecific adsorption effects

O'Carra, Pádraig; Barry, Standish; Griffin, Tadhg

doi:10.1016/0014-5793(74)80993-2

Cited by 76 publications

(24 citation statements)

References 15 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Thus, the spacer arm should ideally be between 9 and 14 Å. This conclusion is in broad agreement with earlier studies carried out with immobilized cofactor derivatives in the non-locking-on mode (2,32,33). It has been suggested that undue lengthening of the spacer arm can result in masking or occlusion of the immobilized ligand due to the ligand-arm assembly adopting a folded conformation (2, 32), but this has not been evident in our studies with the 19.5 Å spacer arms depicted in Figs.…”

Section: Spacer Arm Length and Steric Hindrancesupporting

confidence: 87%

“…In their earlier studies on bioaffinity chromatography, O'Carra and co-workers reported that nonbiospecific interference due to electrostatic interactions can usually be counteracted by maintaining a high salt concentration in the irrigant (2, 26 -29, 34), while a reinforcement of weak adsorption can be achieved by the introduction of marginal nonbiospecific interactions (termed compound affinity-see 28,33,34). Although our later studies have generally supported this, we have identified one exception: 0.5 M KCl abolishes the biospecific locking-on of bovine liver GDH with glutarate to S 6 -linked immobilized NAD ϩ derivatives (56).…”

Section: Nonbiospecific Interference Due To Electrostatic Interactionsmentioning

confidence: 98%

See 1 more Smart Citation

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Mulcahy

O’Flaherty

2001

Analytical Biochemistry

View full text Add to dashboard Cite

Section: Spacer Arm Length and Steric Hindrancesupporting

confidence: 87%

Section: Nonbiospecific Interference Due To Electrostatic Interactionsmentioning

confidence: 98%

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Mulcahy

O’Flaherty

2001

Analytical Biochemistry

View full text Add to dashboard Cite

“…In contrast it is completely retained on agarose-hexane and cannot be recovered with up to I M KC1. These opposite behaviours indicate that the non-specific adsorbing capacity of the hexyl spacer arm [20,21] is obliterated by the covalent attachment of AMP at the extremity of the spacer.…”

Section: Resultsmentioning

confidence: 99%

Affinity Chromatography on Agarose-hexyl-adenosine-5'-phosphate of Methionyl-tRNA Synthetase from Escherichia coli. Application of the Couplings between the Methionine and ATP Sites

et al. 1977

View full text Add to dashboard Cite

Recent studies by us [Biochemistry (1977) 16, 2570-25791 have shown that L-methioninol, a methionine analog lacking the carboxylate negative charge, enhances the affinity of AMP for methionyl-tRNA synthetase while L-methionine antagonizes the nucleotide binding.Such couplings between ligands of the enzyme have now been applied to affinity chromatography of methionyl-tRNA synthetase on an agarose-hex~l-adenosine-5'-phosphate gel (the spacer is attached to AMP at the adenine C-8 position). Retention of the enzyme on this gel column was shown to be dependent on the presence of appropriate concentrations of magnesium and of L-methioninol in the equilibration buffer. The enzyme was then specifically recovered from the column by omitting the amino alcohol or by adding an excess of L-methionine which antagonizes the cooperative effect of L-methioninol.This approach has provided the basis for a new purification procedure of methionyl-tRNA synthetase which leads to a 200-fold purification in a single chromatographic step. In this manner, after 30 -50% ammonium sulfate fractionation of extracts of Escherichiu coli EM 20031 (carrying the F32 episome), 0.25mgxmethionyl-tRNA synthetase was obtained at 90% purity per ml of agarose-hexyI-adenosine-5'-phosphate gel.Several affinity chromatographic methods have already been reported in the case of aminoacyl-tRNA synthetases. These methods are based on the immobilization of either the specific tRNA [l -51, amino acid [6] or aminoalkyl-adenylate [7]. Recently a method for the purification of isoleucyl-tRNA synthetase on blue dextran-Sepharose has been described [8] which is assessed to be affinity chromatography [9].A new approach is now reported which is based upon the couplings (i.e. the change in the dissociation constant of one ligand upon ligation of another ligand) between ligands within the adenylating sites of Escherichiu coli activating enzymes [lo-141. In the case of methionyl-tRNA synthetase, the ligand may couple either synergistically or antagonistically within the enzymic site [14]. For example, L-methioninol, an L-methionine analog lacking the carboxylate function, enhances the binding of AMP while L-methionine itself antagonizes this binding. An abstract of this work was presented at the 10th InternationalEnzyme. Methionyl-tRNA synthetase or L-methionine: tRNAMe' Congress of Biochemistry (Abstract 03-2-1 13).ligase (AMP forming) (EC 6.1.1.10).Such couplings have now been demonstrated to apply to affinity chromatography of methionyl-tRNA synthetase. The AMP nucleotide is immobilized on agarose via the C-8 of the adenine and a six-carbon spacer (hexane) [15]. Substitution of the nucleotide at this position is known not to obliterate binding to the enzyme [12,16]. Methionyl-tRNA synthetase may be specifically bound to the immobilized AMP molecule in the presence of L-methioninol and magnesium in the equilibration buffer. Selective recovery of the enzyme is then achieved by addition of L-methionine. MATERIALS AND METHODSHomogeneous native and trypsin-modified methio...

show abstract

“…It should be noted however, that the present model does not take into Sepharose 4B maximum binding achieved at n = 12-13; decreased binding n > 13 [42] Sepharose 4B non-specific adsorption at low ionic strength [41] N = N consideration the possibility of a deeply buried binding site in the globular protein, which might be expected to increase the value of n at the onset of the plateau region. Similarly, it is to be expected that separations involving shallow or accessible binding sites may not show increased efficiency at increased spacer length even for n < 12.…”

Section: Surntiiury Of the Literaturementioning

confidence: 99%

Sepharose 4B as a Matrix for Affinity Chromatography

Aplin

Hall

1980

European Journal of Biochemistry

View full text Add to dashboard Cite

Nitroxide spin labels were attached to CNBr-activated Sepharose 4B directly and through oligoglycines and w-amino-carboxylic acids of varying length. The homogeneity of the carbohydrate environments of directly attached labels was investigated by measuring dipolar interactions between nitroxides as a function of solvation and of spin dilution with a diamagnetic analogue, as well as by electron exchange between the nitroxides and paramagnetic metal ions in solution. Only the exchange experiment revealed any inhomogeneity, suggesting that a small proportion of sites may be less accessible than the majority. The distances between sites were sufficiently small to allow, in principle, multiple-site interactions between quite small proteins in solution and immobilized ligands. Reorientation of the label at the matrix, characterized by the correlation time z, became more rapid with increasing spacer length n. For n > 12, the decrease in z was less pronounced. The two types of spacer behaved similarly. Thus an ideal spacer length for affinity separations is 12 atoms; this is in good agreement with data from a variety of affinity separations. The results of electron spin resonance studies of the effect of non-aqueous solvent on directly and indirectly labelled Sepharose 4B were used to suggest reasons why enzymes immobilized on Sepharose may be stabilized to denaturing solvents. (Fig. 1 A) i.e. that fraction of agar having fewest charged residues, which is most widely used in its commercially available form of Sepharose gel beads [ll]. Recent X-ray diffraction studies [12] have shown that films and fibres of agarose contain regions of double-helical chain association (junction zones) ; the gel form, however, is thought [13 -171 to contain higher-order aggregates, perhaps of 7 -11 helices. In chemical modificaAbbreviations. ESR, electron spin resonance; HtN-Me4Pip0, 4-amino-2,2,6,6-tetramethylpiperidine-l-oxyl; HzN-Me4Pyr0, 3-amino-2,2,5,5-tetramethylpyrrolidine-l-oxyl; 2,6, tion of the beads, although each agarobiose unit has four hydroxyl groups, the degree of substitution under aqueous conditions never approaches theoretical limits (except perhaps in the presence of very strong base), presumably because of the involvement of many hydroxyls in inter-chain hydrogen bonding, or their substitution by small amounts of sulphate, pyruvate and methyl. The most efficient, and most commonly used, method of immobilising ligands to Sepharose is the cyanogen bromide activation procedure [18,19], which is thought to proceed mainly as shown in Fig. 1 C [20]. An important extra factor shaping the chemistry of affinity chromatography has been the discovery of the need for a spacer arm to separate the ligand from the matrix [21,22] in order to maximise the efficiency of separation or, in other words, the strength of binding between immobilized ligand and solution macromolecule. In the present paper we report a study of Sepharose 4B using nitroxides as model ligands in combination with common chemical methods, i.e. CNBr activation an...

show abstract

Spacer arms in affinity chromatography: Use of hydrophilic arms to control or eliminate nonbiospecific adsorption effects

Cited by 76 publications

References 15 publications

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Kinetic Locking-On Strategy and Auxiliary Tactics for Bioaffinity Purification of NAD(P)+-Dependent Dehydrogenases

Affinity Chromatography on Agarose-hexyl-adenosine-5'-phosphate of Methionyl-tRNA Synthetase from Escherichia coli. Application of the Couplings between the Methionine and ATP Sites

Sepharose 4B as a Matrix for Affinity Chromatography

Contact Info

Product

Resources

About