Measuringthe Absorbanceof Enzyme Activity

... monophopsphate Fig. 2 Equation of Michaelis-menten Enzyme assays are divided into two groups fixed-timed (discontinuos assays), and continuously time. A fixed time assay method is about calculated the waste substrate or product, which produced after fixed time period by using colorimetric chemical method. It is necessary to kept time period as short as possible. The assay was within the linear part of curve. Another assay, which called continuously assay was all about measured the change in substrate or product and this method carried out by the time. The initial velocity provided by setting a tangent to the curve at zero time and recorded its slope (Reed et al 2007). In these experiments, colorimetric assay has been used. This assay is used with artificial substrate produce coloured product which used for detection and quantification of enzyme. Particularly, artificial substrate used when no suitable spectrometric assay was avaible for natural substrates (Reed et al 2007). In spectrophotometric assay this method is applied substances and products absorbs visible or UV light and change in wavelength (Glencross, Ahmed Wang Biomedical science practice). Alkaline phosphatase (AP) is used in this experiment, which produce coloured product. In experiment 1, calculated the initial rate of alkaline phosphatase reaction by the existence of substrate. Stock enzyme activity is calculated in UL (International Units per litre) this calculation is supplied the relationship between absorbance and concentration, which shown in calibration curve (Fig 3) (Laboratories 1 and 2 Schedule). One U determined as how much enzyme that catalyzed the transformation of 1 micro mole of substrate per minute under specific conditions. Substrate that converted in mole divided by time in seconds equals to enzyme activity which unit of katal thus one katal 6x107. (Reed et al 2007)