- 3 11 Self Assessment Questions 10 Points Are Equivalent To 10 Marks In This Project You Are Measuring The Activity Of 1 (188.81 KiB) Viewed 16 times
3.11 Self-assessment
questions (10 points are equivalent to 10 marks) In this project, you are measuring the activity of alkaline phosphatase by measuring the amount of colored product (4-nitrophenol) that appears in a measured time. For this exercise you need to use some data that you have generated. Page | 27 For a reaction that you have carried out, you need to know the absorbance (Abs) after a time in minutes (t): 1. Calculate the rate of color development in Abs min-¹: (1 point) 2. However, rates of reaction are not normally expressed in Abs min-¹, but as an amount of product per minute. You have been given the molar extinction coefficient of 4-nitrophenol (13700 M-¹cm-¹) (1 point) 3. What is the reason for including the 'cm-1' term in the extinction coefficient? (1 point) 4. Use the molar extinction coefficient to convert the absorbance value to a molar concentration (M; mol L-¹). The rate of reaction is now expressed as M min-¹ or mol L₁¹ min 1. (1 point) 5. You will probably find that the number of mol L₁1 min¹ is very small. Thus, you should convert it to μmol L1 min-1. The rate is thus: (1 point) 6. You might alternatively want to express this as the total amount of product produced in your reaction mixture. In this case, the number in 4 needs to be scaled to the volume of reaction plus any stop buffer added. So, the product formation in µmol min-¹ is: (1 point) 7. Often you might want to compare different experiments carried out with different preparations of enzyme, for example, two different cell extracts. In this case it is helpful to calculate the specific activity. This is the enzyme activity, expressed as mmol or μmol of Student name: Student ID: Group number: BIO202 - Advanced Biochemical Methods / Semester 2 / 2021-2022 product per mg of enzyme per minute (mmol mg-¹ min-¹ or µmol mg-¹ min-¹). In other words, this is the activity in 5 divided by the amount of protein used in mg. If you added.......……………….μµl of 100 µg mL-1 alkaline phosphatase to your reaction, then the specific activity of that preparation is: (2 points) 8. Sketch a plot of alkaline phosphatase activity against 4-nitrophenol phosphate concentration. Give a brief explanation for the basis of this relationship. (2 points) Page 28
Experiments to be performed ● Enzyme to be used: calf intestine alkaline phosphatase but found in high concentrations in the bone, intestinal mucosa and in the kidney OH +H₂O T Ox0² + + H+ alkaline phophatase 4-nitrophenyl phosphate 4-nitrophenol Yellow Absorption maximum: 410 nm molar extinction coefficient is 13,700 M-1 cm-1 3 +
What are the components in the reaction? OH +H₂O + H+ alkaline phophatase 1 mM 4-nitrophenol phosphate in 0.1 M Tris, pH Substrate: 9.0 Buffer: 0.1 M Tris, pH 9.0 Free enzyme: 100 μg mL-1 in 0.1 M Tris, pH 9.0 (Immobilised enzyme: 50% (w/v) suspension of agarose beads in 0.1 M Tris, pH 9.0) Stopping solution: 0.1 M sodium phosphate, pH 11.0 Volume of the reaction: 2 ml 4
1 2 3 4 5 6 7 8 9 10 11 Substrate conc vs. OD Free enzyme Substrate Ab (OD) concentration (mm) 0 0 0.1 0.581 0.2 0.797 0.3 0.915 0.4 1.03 0.5 1.044 0.6 1.068 0.7 1.063 0.8 1.07 0.9 1.071 1 1.078 [4-NP] Immobilized enzyme Substrate Ab (OD) concentration (mm) 0 0 0.208 0.256 0.289 0.293 0.252 0.294 0.377 0.425 0.49 0.677 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 12
Time course experiment Free enzyme Immobilized enzyme Time (min) Ab Ab 0.182 1 0.352 0.373 0.539 2 0.405 0.661 3 0.434 0.791 4 0.46 0.903 1.03 5 0.505 1.148 6 0.549 1.22 1.358 7 0.626 1.412 0.64 1.495 0.744 1.509 1.537 10 0.753 1.583 Time (min) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
pH OD (immobi) OD (free) Effect of pH 3.4 4.7 7 9 0.0648 0.0245 0.7484 0.6167 0.2957 0.099 0.6134 0.1453 10.79 0.616 0.261
Effect of temperature OD OD OD 0.444 0.415 0.386 0.408 0.546 0.488 0.532 0.56 0.561 0.489 0.508 0.481 0.478 0.447 0.427 Temp (°C) 15 25 37 45 60
Effect of phosphate concentration OD OD OD 0.589 0.66 0.594 0.597 0.636 0.606 0.654 0.598 0.597 0.576 0.574 0.559 0.56 0.572 0.58 0.607 0.582 0.512 [Sodium phosphate] 10nM 100nM 1uM 10uM 100uM 1mM