Suliman Abdalla.I Ali
- An enzyme assay must be considered valid to ensure accurate data and calculations. In order for an enzyme assay to be valid, it must meet multiple requirements:
- Initial rates are being measured. v0 must be both reproducible and dependent on [E]t. Measuring initial rates demonstrates whether or not the product has reached a substantial concentration. Valid assay conditions exist if the product has consumed less than 5% of the substrate.
- pH is constant: Enzymes have specific pH ranges of activity due to structural sensitivity to proteins which cause enzymes to be sensitive to pH changes. A prepared buffer solution is used to keep the pH constant so that the pH resist changes. Good buffers do not cross membranes; do not absorb light; are chemically stable; and are biochemically inert.
- V0 is proportional to the concentration of the enzyme, [E]t. An assay is only valid when a plot of V0 vs [E]t is linear because the enzyme must be the only limiting factor to the substrate concentration. Additionally, when V0 and [E]t are proportional, the presence of effectors are measurable and one can determine if an inhibitor is competitive, noncompetitive, or uncompetitive.
- V0 is corrected for non-enzymatic conversion: V0(corrected) = V0(+enzyme) – V0(-enzyme). A control factor must be measured while conducting enzyme assays in order to ensure accurate calculations. For non-enzyme controls, buffers are used in place of enzymes
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