Indigogenic substrates for detection and localization of enzymes

Abstract

Indoxyl esters and glycosides are useful chromogenic substrates for detecting enzyme activities in histochemistry, biochemistry and bacteriology. The chemical reactions exploited in the laboratory are similar to those that generate indigoid dyes from indoxyl-beta-d-glucoside and isatans (in certain plants), indoxyl sulfate (in urine), and 6-bromo-2-S-methylindoxyl sulfate (in certain molluscs). Pairs of indoxyl molecules released from these precursors react rapidly with oxygen to yield insoluble blue indigo (or purple 6,6'-dibromoindigo) and smaller amounts of other indigoid dyes. Our understanding of indigogenic substrates was developed from studies of the hydrolysis of variously substituted indoxyl acetates for use in enzyme histochemistry. The smallest dye particles, with least diffusion from the sites of hydrolysis, are obtained from 5-bromo-, 5-bromo-6-chloro- and 5-bromo-4-chloroindoxyl acetates, especially the last of these three. Oxidation of the diffusible indoxyls to insoluble indigoid dyes must occur rapidly. This is achieved with atmospheric oxygen and an equimolar mixture of K(3)Fe(CN)(6) and K(4)Fe(CN)(6), which has a catalytic function. H(2)O(2) is a by-product of the oxidation of indoxyl by oxygen. In the absence of a catalyst, the indoxyl diffuses and is oxidized by H(2)O(2) (catalyzed by peroxidase-like proteins) in sites different from those of the esterase activity. The concentration of K(3)Fe(CN)(6)/K(4)Fe(CN)(6) in a histochemical medium should be as low as possible because this mixture inhibits some enzymes and also promotes parallel formation from the indoxyl of soluble yellow oxidation products. The identities and positions of halogen substituents in the indoxyl moiety of a substrate determine the color and the physical properties of the resulting indigoid dye. The principles of indigogenic histochemistry learned from the study of esterases are applicable to methods for localization of other enzymes, because all indoxyl substrates release the same type of chromogenic product. Substrates are commercially available for a wide range of carboxylic esterases, phosphatases, phosphodiesterases, aryl sulfatase and several glycosidases. Indigogenic methods for carboxylic esterases have low substrate specificity and are used in conjunction with specific inhibitors of different enzymes of the group. Indigogenic methods for acid and alkaline phosphatases, phosphodiesterases and aryl sulfatase generally have been unsatisfactory; other histochemical techniques are preferred for these enzymes. Indigogenic methods are widely used, however, for glycosidases. The technique for beta-galactosidase activity, using 5-bromo-4-chloroindoxyl-beta-galactoside (X-gal) is applied to microbial cultures, cell cultures and tissues that contain the reporter gene lac-z derived from E. coli. This bacterial enzyme has a higher pH optimum than the lysosomal beta-galactosidase of animal cells. In plants, the preferred reporter gene is gus, which encodes beta-glucuronidase activity and is also demonstrable by indigogenic hist...

Localization of Islet-1–Positive Cells in the Healthy and Infarcted Adult Murine Heart

“…Nevertheless, we observed 10 cells in the subendocardial infarct region of 1 heart 7 days after myocardial infarction that showed an atypical but nuclear LacZ staining (Online Figure VII, A). We could not definitely discriminate between a true positive signal and false positivity (eg, caused by macrophages that are able to catalyze X-gal 32 ). Although it might be possible that Islet-1 was expressed between the analyzed time points, the long half-life of β-gal protein of ≈30 hours 33 render it unlikely that such transient expression would go unnoticed.…”

A conditional mouse model for human MUC1-positive endometriosis shows the presence of anti-MUC1 antibodies and Foxp3+ regulatory T cells

“…Mice were sacrificed 72 hours post-AdLacZ injection and the organs of the gynecologic tracts (ovaries, oviducts, uteri) were harvested for X-gal staining of intact tissue (whole mounts) (Kiernan, 2007). The organs were fixed for 5 minutes at room temperature in a fixative solution (2% formaldehyde, 0.25% glutaraldehyde in PBS) and then washed three times with PBS.…”

The progress in the cholinesterase quantification methods

“…The general enzyme constants (Michaelis constant and maximum reaction velocity) were calculated and compared with the standard constants obtained by Ellman photometry. Furthermore, this method seems to be suitable for AChE reactivators in vitro efficacy determination [86] and histochemical enzyme localization [87].…”