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Biochemistry

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Roles in an Enzyme Assay

BIOCHM-P2HGJO

Ornithine transcarbamylase catalyzes the reaction shown.

$$\text{ornithine + carbamyl phosphate} \ \xrightarrow [ ]{ } \ \text{citrulline}$$

In order to assay this enzyme, the enzyme is mixed with 0.1 M Tris, pH 8.5, 0.1 M ornithine pH 8.5 and 0.2 M carbamyl phosphate for 20 minutes. At that time, 10% perchloric acid is added.

In a second step, a portion of this is removed and a mixture is added, boiled, and a purple color is formed that can be detected based on its absorbance at 540 nm.

In an enzyme assay, the activity is measured in units and the value of 1 unit is

.

In the enzyme assay for ornithine transcarbamylase, the role of the 0.1 M Tris pH 8.5 solution is

, and the 0.1 M ornithine pH 8.5 is added to

.

Since this assay required a second step to produce the purple color, this enzyme assay is an example of

.