Conjugation-Ready HRP Maleimide

Conjugation-Ready HRP Maleimide
Horseradish peroxidase (HRP) is commonly used as the signal reporter enzyme in ELISA-based assay formats. Older methods which utilized glutaraldehyde or periodate oxidation-derived Schiff base formation, have their drawbacks (1-2) Glutaraldehyde conjugation methods are difficult to control as they often lead to excessive cross-linking events that lead to protein precipitation (3). Periodate oxidation of the carbohydrate moiety of HRP can be a reasonably successful conjugation method (2). Unfortunately, the oxidation step necessary to form aldehydes on the HRP molecules can also lead to enzyme inactivation and this Schiff base linkage must be properly reduced and stabilized in order to form a permanent covalent bond between the HRP and the protein that is being labeled.

Utilization of maleimide activated HRP (HRP-M) to facilitate the HRP conjugation process enables you to avoid potential HRP enzyme inactivation associated with periodate oxidation procedures, while still controlling the HRP to target protein labeling ratios. This 2-step approach eliminates the risk of uncontrolled cross-linking reaction events that lead to large protein aggregation and subsequent protein precipitation, which is a common occurrence when using glutaraldehyde techniques.

Maleimide conjugation linkers are quite stable and specific for reaction with free SH groups on a target protein/peptide when the reaction is carried out between pH 6.5 and 7.5 (3-4), forming a stable thioether linkage. The excellent hydrolysis stability characteristic in the pH 6.5-7.5 range may be due to the fact that the maleimide ring structure is not directly linked to the aromatic cyclohexane ring (5). At more alkaline pH reaction conditions (>8.0), the maleimide functional groups gradually begin to hydrolyze at a faster rate, into the non-reactive maleamic acid form.

References

1. Avrameas, S. Coupling of enzyme to proteins with glutaraldehyde. Immunochemistry 6, 43-52, (1969).
2. Nakane, P.K., and A. Kawaoi. Peroxidase-labeled antibody. A new method of conjugation.  J. Histochem. Cytochem. 22, 1084-1091, (1974).
3. Hermanson, G.T. “Bioconjugate Techniques”, Academic Press, San Diego, CA. (1996).
4. Smyth, D.G., O.O. Blumenfeld, and W. Konigsberg. Reaction of N-ethylmaleimide with peptides and amino acids. Biochem. J. 91, 589, (1964).
5. Ishikawa, E., M. Imagawa, S. Hashida, S. Yoshitake, Y. Hamaguchi, and T. Ueno. Enzyme-labeling of antibodies. J. Immunoassay 4, 209-327, (1983).