Ephraim Katchalski-Katzir

Use of monoclonal antibodies to detect conformational alterations in lactate dehydrogenase isoenzyme 5 on heat denaturation and on adsorption to polystyrene plates

A monoclonal antibody (mAb) MF30 (IgGl) against human lactate dehydrogenase isoenzyme 5 (HLDH5) was prepared. MF30 was found to bind with high specificity to HLDH5 when the enzyme was adsorbed onto a polystyrene plate but did not recognize the isoenzyme when in solution. The isoenzymes HLDH1, HLDH2 and HLDH3 adsorbed onto polystyrene were not recognized by mAb MF30. Heat-treated HLDH5 (heated at 70 degrees C, pH 7.5 for 45 sec) behaved towards MF30 in the same way as the untreated isoenzyme, i.e. interaction between them took place only after the denatured isoenzyme had been adsorbed onto an ELISA plate. A second mAb, designated 2/66, prepared against porcine lactate dehydrogenase isoenzyme 5 (PLDH5), was found to interact with the porcine isoenzyme when in solution as well as when adsorbed onto polystyrene. However, no such interaction occurred after the isoenzyme had been subjected to heat treatment as above. The mAb 2/66 was found to cross-react fully with the human isoenzyme HLDH5 both in solution and when adsorbed onto polystyrene; however, as in the case of the porcine isoenzyme, all such recognition was lost upon heat denaturation. The above findings suggest that the adsorption of HLDH5 onto a polystyrene surface is accompanied by a conformational change. Denaturation of the enzyme by heat seems to lead to the appearance of a conformation differing in its antigenic pattern from that of the adsorbed enzyme. The data suggest that the mAbs MF30 and 2/66 recognize two different antigenic sites of HLDH5. The antigenic site which is recognized by 2/66 and is present in the native enzyme both when in solution and when adsorbed onto polystyrene disappears on heating. The other antigenic determinant is recognized by mAb MF30 when the enzyme is adsorbed onto a polystyrene surface either before or after heat treatment. This study illustrates the way in which appropriate mAbs might possibly be used as probes for the detection of conformational alterations occurring in proteins under various conditions.

Use of monoclonal antibodies for the preparation of highly active immobilized enzymes.

Publisher Summary This chapter describes a novel method for the preparation of highly active immobilized enzymes. The method is based on the binding of enzymes to suitable carriers through monoclonal antibodies that bind to the enzyme with high affinity without affecting its catalytic activity. The cell fusion technique developed by Kohler and Milstein makes it possible to prepare a great variety of monoclonal antibodies directed toward specific, well-defined antigenic sites of a protein. The available data suggest that for many enzymes it might be possible to prepare corresponding monoclonal antibodies that bind to them with high affinity without affecting their catalyic activity. Binding of such antibodies to insoluble carriers should thus yield carrier-monoclonal antibody conjugates that bind readily with the corresponding enzymes to yield highly active immobilized enzymes. In such immobilized enzyme preparations, the enzyme is bound through a specific, preselected, well-defined site of the enzyme.

Interaction of carboxypeptidase A with monoclonal antibodies.

Several mouse monoclonal antibodies to carboxypeptidase A (CPA) were prepared and purified, and their interaction with the enzyme was investigated. CPA is a well-characterized zinc-containing exopeptidase exhibiting peptidase as well as esterase activity. The antibodies obtained could be classified as follows: antibodies inhibiting mainly the peptidase activity of the enzyme, antibodies inhibiting mainly its esterase activity, antibodies affecting both activities, and antibodies which bind to the enzyme but have no marked effect on its catalytic properties. Binding constants of approximately 10(6) M-1 were obtained for most of the antibody-enzyme complexes tested. Additional information on the effect of the monoclonal antibodies on the active site of CPA was obtained by determining the change in the circular dichroism spectra of arsanilazotyrosine-248 carboxypeptidase A occurring as a result of the interaction of the enzyme with the antibodies studied. These findings suggest that CPA possesses at least three different specific antigenic sites, and that the active site of the enzyme for its peptidase activity differs from that for its esterase activity, though both sites seem to overlap to a considerable extent.

Use of monoclonal antibodies in the detection of structural alterations occurring in lysozyme on heating

Seven murine anti-hen egg-white lysozyme (HEL) monoclonal antibodies (MAbs), which recognize distinct epitopes of the native enzyme, were used as macromolecular probes to detect structural or conformational alterations occurring in HEL on heating at 95 degrees C, pH 5. As the interactions of the heat-treated HEL with its corresponding MAbs were carried out at room temperature, only irreversible structural and/or conformational alterations could be detected. The transformation of the native enzyme into its denatured form was followed electrophoretically and chromatographically. The denatured enzyme was more negatively charged at pH 8.4 and exhibited a longer retention time on reverse-phase HPLC than native HEL. Its specific catalytic activity was considerably lower than that of the native enzyme. Of the seven MAbs tested in competitive ELISA assays with native and heat-treated HEL only one, MAb D74.3, failed to recognize the heat-treated enzyme. This antibody, which is directed toward the active site region of the enzyme, was ineffective in inhibiting the catalytic activity of the heat-treated HEL using M. lysodeikticus as substrate. In contrast, the monoclonal antibody D1.3, which recognizes an epitope remote from the active site of HEL, inhibited the catalytic activity of the native as well as the heat-treated enzyme. The results indicate that the active site of HEL undergoes an irreversible structural alteration on heating for 2 hr at 95 degrees C, pH 5. No irreversible structural changes could be detected in the other regions of HEL recognized by the corresponding MAbs.

Design and Synthesis of Organic Molecules Based on Molecular Recognition

A great deal of work has been carried out by chemists, physicists and physical chemistry in order to gain a better understanding of the principles determining specific molecular interaction [1–3]. The biologist, of course, is well aware of specific biological reactions in practically every field of study in which he is involved. Specific biological reactions occur in many of the vital life processes, such as the interactions between enzymes and their corresponding substrates and inhibitors, between hormones and their receptors, between antibodies and antigens, and between cell and cell. Powerful physical techniques such as X-ray and NMR have made it possible to determine on an atomic level the conformation of some important biopolymers — enzymes, antibodies and nucleic acids — and to shed new light on the atomic structure of the molecular domains responsible for the specific interactions exhibited by these macromolecules. Furthermore, analysis of the conformation of specific biopolymer-ligand complexes, using the above techniques, has opened up new possibilities for the evaluation of the forces and energetics involved, and led to the elucidation of some of the molecular mechanisms that characterize biocatalysis.