Spectrophotometric Assay

Spectrophotometric Assay of Polyphenol Oxidase

1. Introduction

Polyphenol oxidase is a natural enzyme present in banana. Also found in banana is dopamine, when these bind together they form dopamine quinone, which is spontaneously oxidised to other quinones, first of which is red quinone.

2. Method

As per protocol

3. Results

| Table 1: Volume of banana extract (µl) and their corresponding absorbance readings (AU) |

Time (minutes) | 10 (µl) | 20 (µl) | 30 (µl) | 40 (µl) |

0.170 | 0.035 | 0.094 | 0.173 | 0.193 |

0.330 | 0.068 | 0.160 | 0.254 | 0.289 |

0.500 | 0.099 | 0.211 | 0.313 | 0.343 |

0.670 | 0.133 | 0.269 | 0.363 | 0.373 |

0.830 | 0.163 | 0.312 | 0.402 | 0.390 |

1.000 | 0.196 | 0.344 | 0.433 | 0.395 |

4. Calculation Example

For the enzyme activity of 20 µl of banana extract

We used the beer lambert equation A = ε c l where A is absorbance (gradient of line on graph), ε is extinction coefficient (2.515) and l is the path length.

The equation was rearranged to make C the subject

C = A / (ε x l)

C = 0.4125 / (2.515 x 1)

= 0.164 mM per minute

0.164 x 3/1000 = 4.92 x10-4

4.92 x10-4 x 103 = 0.492

0.492 x 50 = 24.6 µmoles per minute per ml enzyme

Table 2: The calculated enzyme activity for each volume of banana extract |

Enzyme activity (µmol min-1, mL-1 ) | volume of banana extract (µl) |

24.60 | 10 |

24.60 | 20 |

15.10 | 40 |

8.95 | 80 |

5. Discussion

Enzymes are protein catalyst that speeds up the rate of a reaction, by providing an alternative route for the same reaction that has lower activation energy.

Alexander et al (2011) suggested that many enzymes require extra entities binding to them to activate, extra entities such as co- enzymes, (which accept or donate functional groups to assist in the enzymatic reaction).

Each assay of the different volumes of banana extract did not produce the same enzyme activity in this...