I am working on enzyme immobilization. can anyone suggest me
How much volume of enzyme and substrate should i take to check the enzymatic activity of immobilized enzyme through UV. Thanks
Although the question seems a bit vague as the final volume of the reaction mixture depends on the size of the cuvette that you wish to use in a spectrophotometer. The cuvette size may be as small as 100 micro litres or 3 mL. even micro-drop spectrophotometers are available now!
Hello Kamaran, Shamsher has addressed the first part. What I can add is that the concentration of substrate will influence the rate of reaction but there are several factors to consider in selecting the ‘right’ concentration. From a practical point of view one key consideration is the amount of product that must be generated in order to give a measurable assay signal. Since the rate of an enzyme reaction is likely to fall when more than about 15% of the substrate has been hydrolysed, the initial concentration of substrate should generally be at least 10x the concentration of product that is known to give an acceptable assay signal. Additionally, the Michaelis-Menten equation is useful in that it helps you to select a suitable concentration of substrate if the Km is known. So, consider the Km for the substrate. While adding more substrate generally means that you will see higher activity values, the relationship is not linear and the cost of the substrate may be an issue.
The signal for most enzyme assays is proportional to the assay volume and attempts to miniaturise (e.g. to conserve reagents) will usually lead to lower signals. However, absorbance assays are often an exception, and a switch from a 3ml cuvette to 1ml micro-cuvette, for example, will not change the absorbance reading if the width (path length) of the cuvette is still 1 cm (i.e. the light still passes through the same ‘length’ of liquid). This is because absorbance is proportional to path length, not to the volume of sample.
-Enough volume so that your light beam goes through the liquid is the answer to your Question.
-Maurice probably answered what you wanted to ask.
-As you write you are working with immobilised enzyme keep in mind that your enzyme carrier (most likely) will scatter the light and your absorbance measurement will be complicated. End-Point measurements filtering of the immobilised enzyme or spinning your sample taking the super natant for analysis are methods to circumvent this.
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