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Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation for Advanced Student Experiments
https://kjunshin.repo.nii.ac.jp/records/2000177
https://kjunshin.repo.nii.ac.jp/records/2000177c158dba9-38d3-4c73-ae4a-f0586eeae882
| 名前 / ファイル | ライセンス | アクション |
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Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation for Advanced Student Experiments.pdf (1.8 MB)
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| Item type | 紀要論文 ／ Departmental Bulletin Paper(1) | |||||
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| 公開日 | 2025-04-23 | |||||
| タイトル | ||||||
| タイトル | Measurement of Alkaline Phosphatase Activity Based on Absorbance and the Michaelis-Menten Equation for Advanced Student Experiments | |||||
| 言語 | en | |||||
| 言語 | ||||||
| 言語 | eng | |||||
| 資源タイプ | ||||||
| 資源タイプ識別子 | http://purl.org/coar/resource_type/c_6501 | |||||
| 資源タイプ | departmental bulletin paper | |||||
| 著者名(日) |
南, 勇真
× 南, 勇真 |
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| item_2_description_12 | ||||||
| 内容記述タイプ | Abstract | |||||
| 内容記述 | 【Introduction】Enzymes play a pivotal role in living organisms by catalyzing a variety of essential reactions. It is particularly important for students involved in nutrition or biochemistry to learn about enzymes and their properties from a chemical perspective. On the other hand, it is difficult to perform rigorous measurements of enzyme activity at the student experimental level. This bulletin article presents simple experiments and analyses for understanding the Michaelis-Menten equation. 【Methods】The hydrolysis of phosphate by alkaline phosphatase (ALP) was used as a model reaction. Commercially available ALP from E.coli. was used for the dephosphorylation of p-nitrophenyl phosphate (p-NPP) to p-nitrophenol (p-NP). The reaction progress was monitored by measuring the absorbance at 420 nm derived from p-NP under alkaline conditions. Inorganic phosphate was used as an ALP inhibitor. The concentration of the product (p-NP) was estimated from a calibration curve, and approximate reaction rates were also calculated. Based on Michaelis-Menten equation, Vmax and Km were also obtained in each reaction conditions. All data were analyzed using Excel. 【Results and Discussion】A calibration curve for p-NP was constructed, demonstrating strong linearity based on Lambert-Beer’s law, enabling precise quantification of reaction products. The reaction rate was highest within the first minute and decreased over time, with the maximum concentration of p-NP (100 µM) reached within 15 minutes. The Michaelis-Menten kinetics of ALP were analyzed, yielding Vmax = 0.0254 mM/min, and Km = 0.0290 mM from a Lineweaver-Burk plot. The inhibitory effect of inorganic phosphate (Pi) was assessed by introducing two concentrations of Pi (0.1 and 0.2 mM). Analysis of the Lineweaver-Burk plots showed increased slopes with constant y-intercepts, indicating competitive inhibition. This was consistent with a mode of inhibition in which the inhibitor binds to the enzyme’s active site, increasing Km without altering Vmax. These results provide insights into the kinetic properties of ALP and the competitive inhibition exerted by its reaction byproduct, inorganic phosphate. 【Summary】In this report, protocols for ALP activities measurements were introduced for student experiments. The measurements are based on spectroscopic analysis and Michaelis-Menten equation. These experiments need no special equipment or software and are easy to perform. The Lineweaver-Burk plot of experimental results shows good accordance with representative competitive inhibition. Experiments on other forms of inhibition could be conducted by changing the inhibitor. |
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| 言語 | en | |||||
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| 収録物識別子タイプ | ISSN | |||||
| 収録物識別子 | 2759-050X | |||||
| item_2_relation_13 | ||||||
| 関連タイプ | isVersionOf | |||||
| 識別子タイプ | Local | |||||
| 関連識別子 | - | |||||
| bibliographic_information |
ja : 鹿児島純心大学看護栄養学部紀要 巻 -, 号 29, p. 24-31, ページ数 8, 発行日 2025-03 |
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