Products Capable of Coupling with Diazonium Salts

Many hydrolytic enzymes (e.g., aminopeptidases, esterases, gly-cosidases, phosphatases) are visualized on electrophoretic gels using artificial substrates that are naphthol or naphthylamine derivatives. When naphthol or naphthylamine is liberated enzy-matically, it immediately couples with a diazonium salt. An insoluble colored precipitate (an azo dye) is formed as a result of the coupling reaction in gel areas where specific hydrolase activity is located.

There are two main components of the azo coupling system. The first one is the diazonium ion. The diazonium ions are not stable and need to be in a salt form for prolonged storage.3 A number of different stabilized diazonium salts are now commercially available. Usually these are salts or double salts of zinc chloride (e.g., Fast Blue B salt, Fast Blue BB salt, Fast Blue RR salt, Fast Violet B salt), tetrafluoroborate or sulfate (e.g., Fast Garnet GBC salt), naphthalenedisulfonate (e.g., Fast Red B salt), etc.4 For further stabilization such inert additives as aluminum, magnesium, sodium and zinc sulfates, magnesium oxide and bicarbonate, and others can also be added to commercial dia-zonium salt preparations.

It should be taken into account that some enzymes may be inhibited by these additives. When the diazonium salt acts as an enzyme inhibitor, a two-step, or so-called postcoupling, staining procedure is recommended. In this procedure, the gel is initially incubated in substrate solution for an essential period of time (usually 30 min) and only then diazonium salt is added. However, the one-step azo coupling procedure is usually preferable because it considerably reduces diffusion of enzymatically liberated naphthols and naphthylamines and results in development of more sharp and distinct activity bands.

Dissolved diazonium salts give very unstable ions, so solutions should be prepared immediately before use. The stability of diazonium ions depends on the pH of the staining solution. For example, Fast Black K salt is stable in acidic pH, while Fast Blue RR salt is recommended for use at neutral and alkaline pH. High temperature also contributes to decreasing stability of dia-zonium salts. When the azo coupling system is used to detect the enzyme, a compromise should be achieved between the pH optimum of the enzyme activity and diazonium salt stability. If pH values extreme for diazonium salt stability must be used, it is recommended that the diazonium salt solution be replaced as often as necessary, depending on the total period of gel incubation. Because diazonium ions can themselves act as enzyme inhibitors, the use of optimal concentrations of 1 mg/ml is recommended.3

The second component of the azo coupling system is an enzyme-specific artificial substrate that is an amide, an ester, or a glucoside of the coupling agent (naphthol or naphthylamine). The substrates that are derivatives of substituted naphthol (e.g., 6-bromo-2-naphthol) or substituted naphthylamine (e.g., 1-meth-oxy-3-naphthylamine) may be preferable; this is because some substituted naphthols coupled with diazonium ions give azo dyes of greater insolubility. The use of derivatives of 1-methoxy-3-naphthylamine, which complexes much more quickly with dia-zonium ions than does 2-naphthylamine, reduces the problem of product diffusion during color formation.

Examples of the use of the azo coupling system in electrophoretic zymography are the detection of esterases (3.1.1 ... — EST, Method 1) and acid phosphatase (3.1.3.2 — ACP, Method 3), with a-naphthol as the coupling agent; the detection of P-glucuronidase (3.2.1.31 — P-GUS, Method 2), with naphthol-AS-BI or 6-bromo-2-naphthol as coupling agents; the detection of leucine aminopeptidase (3.4.11.1 — LAP, Method 1), with 2-naphthylamine as the coupling agent; and the detection of some proteinases (3.4.21-24 ... — PROT, General Principles of

Detection), with 1-methoxy-3-naphthylamine as the coupling agent.

Diazonium ions also couple with such enzymatic products as oxaloacetate, which serves as a coupling agent of the azo coupling system in the detection of some nonhydrolytic enzymes, e.g., glutamic-oxaloacetic transaminase (2.6.1.1 — GOT, Method 1), phosphoenolpyruvate carboxylase (4.1.1.31 — PEPC), and pyruvate carboxylase (6.4.1.1 — PC).

The azo coupling system was the first enzyme-staining system successfully adopted from histochemistry for purposes of electrophoretic zymography.5 A great number of zymographic techniques described in Part III are based on the use of this staining system.

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