Padraig O'Carra

Affinity chromatography of lactate dehydrogenase Model studies demonstrating the potential of the technique in the mechanistic investigation as well as in the purification of multi-substrate enzymes

The general potential of affinity chromatography in enzyme isolation and purification is now well recognized [l-3] . No general treatment of the special problems likely to be encountered in applying it to multi-substrate enzymes seems to have been published, nor do some of the special possibilities of the technique for such enzymes seem to be generally recognized. This paper describes model studies on lactate dehydrogenase (LDH) which explore some of these problems and possibilities. The results demonstrate that the effectiveness of affinity chromatography as a purification tool for certain types of multi-substrate enzyme may be greatly increased by taking advantage of kinetic characteristics. It is also shown that other advantages may accrue in that affinity chromatography is capable of yielding very clear-cut information about multi-substrate kinetic mechanisms. For example, the technique clearly demonstrates the compulsory-ordered mechanism of LDH and also indicates that it is the nicotinamide end of NADH which induces the binding site for pyruvate analogues, even though it is the “AMP portion” which is mainly responsible for the binding of the pyridine nucleotide to LDH [4].

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

In previous communications [l-4] we have shown that failure to distinguish adequately between biospecific adsorption (bioaffinity) and nonbiospecific adsorption has seriously affected the validity and usefulness of much published affinity chromatographic work. In some cases nonbiospecific interference is so severe that it eclipses any element of biospecificity in the chromatography. While some of this interference may be attributable to ion-exchange effects (see, e.g., refs. [5-lo]), evidence suggests that the most serious and intractable interference is attributable to the commonly-used, hydrocarbon spacer arms which themselves promote nonspecific adsorption of a wide range of enzymes. This adsorption appears to be associated with the hydrophobic nature of these spacer arms and it was postulated that the interfering, nonspecific adsorption results mainly from hydrophobic interactions between these and the enzymes [ 1,2,4,11 J . It was suggested that replacement of the currently-used arms by less hydrophobic ones might lessen or eliminate @he interference [I] . We describe here the preparation and characteristics of more hydrophilic spacer arms, and we show that the use of such arms in the construction of affinity gels does indeed eliminate most of the interfering adsorption effects. Our previous conclusions regarding the source and nature of the interference are largely verified by these results. 2. Experimental

[8] Interfering and complicating adsorption effects in bioaffinity chromatography

Publisher Summary This β -galactosidase system has played an important role in the development of the view that immobilized ligands with dissociation constants in the millimolar range are capable of promoting very strong specific adsorption, and this view in turn has led to the development of the concept of “progressively perpetuating effectiveness.” This concept is not consistent with much other evidence, or with the theoretical treatment summarized in the next section, and it is difficult to reconcile with kinetic theory in general. Again, the observations that largely gave rise to this concept are explainable in terms of nonbiospecific rather than biospecific adsorption.

Separation and identification of biliverdin isomers and isomer analysis of phycobilins and bilirubin

Abstract One-dimensional thin-layer chromatographic systems are described for the resolution of the IXα, IXβ, IXγ, and IXδ isomers of protobiliverdin and mesobiliverdin, and each isomer is unambiguously identified. The separations may be applied on a preparative scale or as a micro-scale method for isomer analysis of bilverdin preparations or of bilins which can be converted thereto. Besides being very sensitive and reliable, the analytical method also yields much more information than degradativ methods. Applied to some naturally occurriing bilins, it shows that bilirubin from pig bile is about 99.6% IXα with traces of IXδ and of IXβ and IXγ. Phycoerythrobilin and phycocyanobilin seem to be entirely IXα. Mesobiliverdin IXα, prepared from bilirubin by the usual alkaline reduction followed by dehydrogenation, contains two extra minor, seemingly isomeric mesobiliverdins. A new method of preparation, avoiding alkaline reduction, yields only tru mesobiliverdin IXα.

Affinity chromatographic differentiation of lactate dehydrogenase isoenzymes on the basis of differential abortive complex formation

Lactate dehydrogenase (LDH) has a compulsoryordered kinetic mechanism [ 1,2] which requires that NADH must bind to the enzyme before pyruvate can bind (fig. 1, left hand side). The H-type isoenzyme, but not the M-type, is characterized by substrate inhibition at high pyruvate concentrations [3 1. This substrate inhibition has been explained [3] in terms of the formation of a dead-end complex in which NAD+ acts as an analogue of NADH in promoting the binding of pyruvate (fig. 1, branch on right hand side). The resulting enzyme-NAD’-pyruvate complex is commonly termed an abortive complex, because no catalysed electron transfer can occur between the two oxidized substrates (in contrast to the ‘productive’ enzyme-NADH-pyruvate complex). The negligible substrate inhibition exhibited by the M-type isoenzyme suggests that it forms the abortive complex only to a negligible extent compared to

[77] Lactate dehydrogenase: Specific ligand approach

Publisher Summary In the search for an effective immobilized ligand, attention was concentrated on analogs of pyruvate rather than of lactate, because kinetic studies indicate that pyruvate has considerably higher affinity for the enzyme. As explained in this chapter, the kinetic mechanism of the enzyme requires the addition of NADH to the irrigating buffer during affinity chromatography. Under these conditions catalytically susceptible immobilized analogs (e.g., β-mercaptopyruvate, attached through the β-thiol group) underwent enzymatic reduction when the enzyme was applied, and such analogs proved to be unsatisfactory for affinity chromatography. Oxamate is a structural analog of pyruvate that is not catalytically susceptible, but retains the binding characteristics of pyruvate for lactate dehydrogenase. This analog, immobilized through the amide grouping proved to be a very effective affinity chromatographic ligand. The amide nitrogen group corresponds to the methyl group of pyruvate and was chosen as the point of attachment because available specificity studies indicated that pyruvate analogs in which the methyl group is substituted remain effective as substrates for lactate dehydrogenase.

The lactate dehydrogenase of the icefish heart: biochemical adaptations to hypoxia tolerance

Cardiac lactate dehydrogenase from the hemoglobin- and myoglobin-free antarctic icefish has been purified by affinity chromatography. Structural and kinetic properties of the enzyme were found close or identical to those of its skeletal muscle counterpart and other M-type lactate dehydrogenases. A model involving a dual oxidative-anaerobic metabolism of the icefish heart is proposed.

FURTHER STUDIES ON THE BIOAFFINITY CHROMATOGRAPHY OF NAD+ -DEPENDENT DEHYDROGENASES USING THE LOCKING-ON EFFECT

Previous studies have capitalized on ordered kinetic mechanisms in the design of biospecific affinity chromatographic methods for highly efficient purifications and mechanistic studies of enzymes. The most direct tactic has been the use of immobilised analogues of the following, usually enzyme-specific substrates, e.g., lactate/pyruvate in the case of lactate dehydrogenase for which NAD+ is the leading substrate. Such immobilised specific substrates are, however, often difficult or impossible to synthesise. The locking-on strategy reverses the tactic by using the more accessible immobilised leading substrate, immobilised NAD+, as adsorbent with soluble analogues of the enzyme-specific ligands (e.g., lactate in the case of lactate dehydrogenase) providing a substantial reinforcement of biospecific adsorption sufficient to effect adsorptive selection of an enzyme from a group of enzymes such as the NAD(+)-specific enzymes. The value of this approach is demonstrated using model studies with lactate dehydrogenase (LDH, EC 1.1.1.27), alcohol dehydrogenase (ADH, EC 1.1.1.1), glutamate dehydrogenase (GDH, EC 1.4.1.3) and malate dehydrogenase (MDH, EC 1.1.1.37). Purification of bovine liver GDH in high yield from crude extracts is described using the tactic.

Lactate dehydrogenase in plants: Distribution and function

Abstract Contrary to previous reports, lactate dehydrogenase (LDH; EC 1.1.1.27) occurs in all green land plants ranging from flowering plants to mosses. Conditions must be carefully designed and monitored, however, to achieve detection and extraction, and even then the specific activity is always orders of magnitude lower than that typically encountered in animal tissues. Green algae contain even lower levels of an NAD-dependent LDH activity not clearly identified as EC 1.1.1.27. Red and brown algae seem to lack LDH activity entirely. A striking feature of all the flowering plants investigated was a prominent peak of expression of LDH in stem tissue, at, or immediately above, soil level which was paralleled by a similar peak of alcohol dehydrogenase (ADH; EC 1.1.1.1) activity. The possible functional significance of this, and the function(s) of plant LDH generally, are discussed.

Purification and substrate kinetics of plant lactate dehydrogenase

Abstract Lactate dehydrogenase (LDH) from turnip ( Brassica rapa ; Cruciferae), purified to electrophoretic homogeneity using affinity chromatography, has a native M r , of 157 × 1O 3 and a subunit M r , of 38 × 10 3 . The LDH from turnip shows the same relative effectiveness (relative V max and K m values) as the mammalian H 4 and M 4 isoenzymes with pyruvate, lactate and glyoxylate (oxoacetate and dihydroxyacetate) as substrates. All three LDH types show no activity with glycolate (hydroxyacetate). The affinities for these and a range of competitive inhibitory analogues shows a consistent pattern of highest affinity for the H 4 mammalian isoenzyme, medium affinity for the M 4 form and lowest affinity for the plant enzyme, in a ratio of about 10:3:1, respectively. The catalytic mechanism of the plant enzyme is very similar to that of the mammalian forms. The major physiological activity of the plant LDH is considered to be pyruvate reduction, rather than the disproportionation of glyoxylate that has been proposed as a plant cell pH-stat.