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Enzyme Assay, Enzyme Activity and Specific Activity
Inside our body and inside our cells, we have a great variety of different types of proteins. If we want to study a specific type of protein, how do we get ahold of that protein of interest? That is, if we have a sample of solution that contains many different kinds of proteins, how do we know if the protein is in that mixture and if it is in the sample, how do we then purify the sample and isolate that protein? To answer the first part of the question, we need to conduct an assay. A protein assay is some sort of procedure that allows us to determine whether or not the target protein of interest is in the sample. It uses some sort of unique property or functionality of that protein. Whenever we are carrying out an assay, we have to keep in mind two questions - (1) is the target protein in the sample ? (2) If it is, what is the concentration of that protein? To answer the first question, we can measure the enzyme activity of that protein. For instance, lets consider the enzyme lactate dehydrogenase. Lactate dehydrogenase catalyzes the conversion of lactate into pyruvate, in the process also reducing nicotinamide adenine dinucleotide. One property of reduced nicotinamide adenine dinucleotide (NADH) is it has the ability to absorb light with a wavelength of 340 nanometers. This means that if the protein is present in the sample and we mix it with NAD+ and lactate, we should be able to see light being absorbed at that specific value. As more and more protein (NADH) is produced, more and more light should be absorbed. The enzyme activity refers to the number of moles of product formed per unit time. Once we know the enzyme activity and the concentration of the enzyme, we can then determine the specific activity of the enzyme. The specific activity is the ratio of the enzyme activity to enzyme concentration. This quantity can be used to measure the purity of our sample. During the purification process, the specific activity should increase up until we reach a pure sample, at which point the value will remain constant.
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