Phosphatases are enzymes that catalyse the removal of phosphate from other molecules, releasing inorganic phosphate ( $$\(\mathrm{P}_{\mathrm{i}}\)$$ ). A phosphatase can be extracted from mung bean seedlings, Vigna radiata. A student found a published method to extract phosphatase from mung bean seedlings. - Use a pestle and mortar to grind mung bean seedlings, with a small volume of distilled water, to make a paste. - Add distilled water to the paste to make a mixture with a total volume of $$\(50 \mathrm{~cm}^{3}\)$$. - Filter the mixture and put the filtrate into a clean centrifuge tube. - Centrifuge the filtrate until a solid pellet is formed, as shown in Fig. 1.2. - Pour the liquid extract containing phosphatase into a clean test-tube. The student read that the optimum pH for phosphatase extracted from mung bean seedlings is less than pH 7.0 . The student decided to investigate phosphatase activity, using the substrate phenolphthalein phosphate (PPP). In the first four steps of the method, the student: - mixed $$\(5 \mathrm{~cm}^{3}\)$$ of pH 6.0 buffer solution with $$\(1 \mathrm{~cm}^{3}\)$$ of a $$\(1.0 \%\)$$ solution of PPP in a test-tube - added $$\(1 \mathrm{~cm}^{3}\)$$ of the phosphatase extract to the test-tube and started a timer - incubated the test-tube for 10 minutes in a water-bath at $$\(30^{\circ} \mathrm{C}\)$$ - stopped the enzyme reaction after 10 minutes, by adding $$\(5 \mathrm{~cm}^{3}\)$$ of $$\(10.0 \%\)$$ solution of sodium carbonate. Sodium carbonate solution is alkaline. (i) The buffered PPP solution and the phosphatase extract were mixed and then placed in the water-bath. The student identified this as a source of error in the method. Explain why this is a source of error and state how you would modify the method to remove this source of error. explanation . . . . modification . . . . (ii) Suggest how the addition of $$\(10.0 \%\)$$ solution of sodium carbonate stops the enzyme reaction. .

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