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Cytology · Year 1 · Medical University of Sofia

05

Methods for the demonstration of enzymes

Free notes for topic 05 of the Cytology syllabus, open without an account. Written by a senior student against the syllabus question and checked line by line by a second student before publishing. How content is made

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Summary

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Enzyme histochemistry identifies and localises cellular structures by using the enzymatic activity present in those structures.

The key idea is indirect. We cannot observe the enzyme itself. Instead we let the enzyme act on its substrate, then trap the product of that reaction with a coloured marker, and look at where the colour appears. The enzyme is located by the mess it leaves behind.

Two examples carry the topic: acid phosphatase gives a brown colour and identifies lysosomes and the Golgi complex; succinate dehydrogenase gives a blue colour and identifies mitochondria.

1. What enzyme histochemistry is

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Enzyme histochemistry is a method for identifying and localising cellular structures by using the enzymatic activity present in those structures.

The central limitation is this: we cannot observe the enzyme itself. Instead we obtain a visible reaction that provides us with indirect information about the presence of the enzyme.

2. Principles

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The tissue must be fresh and unfixed, and it is sectioned on a cryostat.

The reason is that fixation would destroy the very thing being detected. An enzyme is a protein, and the fixatives and solvents of the paraffin technique denature proteins. Cutting the tissue frozen, on a cryostat, avoids the effects of heat and organic solvents on enzymatic activity.

3. The process

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The method runs in four steps:

  1. The tissue section is immersed in a solution containing the substrate of the enzyme to be localised.
  2. The enzyme acts on its substrate.
  3. The tissue section is put in contact with a marker compound, a dye or a heavy metal, that reacts with the product of the enzyme-substrate reaction.
  4. The final product is insoluble and visible by light or electron microscope, which identifies the enzyme location.

Summarised as two equations:

enzyme + substrate = unstained reaction product

product + dye (marker) = insoluble coloured final reaction product

The word to hold on to in step 4 is insoluble. A soluble product would diffuse away from where it was made, and the colour would no longer mark the place the enzyme was.

4. Examples of enzymes used to identify cellular structures

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Two enzymes are the standard examples, one for each of two organelles.

Acid phosphatase

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Acid phosphatase, demonstrated by the method of Gomori, is used for identifying lysosomes and the Golgi complex.

A brown colour tells us the presence of acid phosphatase. For example, in the kidney there are a lot of lysosomes.

Succinate dehydrogenase

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Succinate dehydrogenase is used for identifying mitochondria.

A blue colour tells us the presence of the enzyme. For example, in muscle cells there are a lot of mitochondria.

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