Non-deleterious inhibition of endogenous peroxidase activity (EPA) by cyclopropanone hydrate: a definitive approach

Endogenous peroxidase activity (EPA) poses a serious problem in immunoperoxidase localization of antigens unable to withstand deleterious effects of aldehyde fixatives, alcohols, and various oxidative reagents. This has forced the development of more selective inhibition methods. Of these, phenylhydrazine or azide combined with small amounts of H2O2 have proved quite effective. However, the precise mechanism of the action of these compounds on EPA generating proteins is not understood. Cyclopropanone hydrate is a compound whose inhibitory action on the heme moiety of horseradish peroxidase is well understood. The aim of this study was to investigate the effect of this compound on EPA and to compare its efficiency with that of optimal phenylhydrazine and sodium azide regimens. In addition, any gross deleteriousness of cyclopropanone hydrate towards immunoperoxidase immunolocalization of three of the most delicate lymphocyte surface antigens was investigated. Cyclopropanone hydrate was found to inhibit EPA with progressing strength between 0.15-15 mM. Over this range, H2O2 was found necessary for inhibition only for cyclopropanone hydrate concentrations up to 0.15 mM. Beyond this amount, the compound inhibited EPA equally strongly in the presence or absence of H2O2, reaching near-maximum inhibition at 15 mM. This and the H2O2-requiring regimens were found to cause no gross diminution in immunoperoxidase staining of CD4, CD6, and CD8 antigens in snap-frozen, acetone-fixed human tonsil sections. Cyclopropanone hydrate therefore provides a definitive non-deleterious mode of inhibiting EPA for immunoperoxidase staining of delicate antigens.