Experiment7-Enz - Experiment 7(Lab Period 8 Quantitative...

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Experiment 7 (Lab Period 8) Quantitative Determination of Phosphatase Activity Phosphatases are enzymes that catalyze the hydrolysis of organic-phosphate compounds, releasing inorganic phosphate from the rest of the molecule. The general reaction is: O O R ? O ? P ? OH ? HOH R ? OH ? HO ? P ? OH OH OH Phosphatases serve a variety of functions in living organisms. Some help to digest food so that the smaller products can be absorbed or metabolized. Such phosphatases occur as secreted enzymes, as in the mammalian intestine. They also occur as dormant enzymes in seeds; during germination, they become active and mobilize stored food to be used by the emerging seedling. Other phosphatases occur in the lysosomes of phagocytic cells and help to digest particulate matter captured during phagocytosis. Still others are active in the cytoplasm and serve to recycle phosphorus in metabolism or to remove phosphate groups from proteins whose activities are regulated by the addition and removal of phosphate groups. Many phosphatases are not very specific with respect to the compounds they use as substrates, and any one phosphatase will often be able to hydrolyze various organo-phosphates. The low specificity will allow us to utilize only one substrate to test the activity of two different phosphatases in this experiment. The test reaction that will be used to measure (assay) enzyme activity is hydrolysis of para-nitrophenylphosphate: OH- ? -nitrophenylphosphate ? ? -nitrophenol ? ? -nitrophenolate (colorless) (colorless) (yellow) In this assay, the reaction is timed from the time of addition of enzyme to the time of addition of strong alkali (0.2 M NaOH). The reaction catalyzed by the enzyme is the first of the two shown in the diagram; this is the reaction we want to measure. Since both compounds are colorless we cannot do so directly, instead we add NaOH. The NaOH has two effects: (l) it converts p-nitrophenol to p-nitrophenolate and develops the yellow color, and (2) it stops enzyme activity. By measuring the quantity of -nitrophenolate we are in effect measuring the quantity of -nitrophenol that was present (the product) before the addition of NaOH. In performing this assay, measure as accurately as possible; the interpretability of your data will depend on the accuracy of your measurements of all the volumes of all the components of the reaction mixtures. The lab that you will perform consists of two parts. The first is a determination of the influence of phosphatase on the rate of the reaction and the second consists of an investigation of the influence of pH on the activity of phosphatase.
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Experiment7-Enz - Experiment 7(Lab Period 8 Quantitative...

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