Experiment 4: Protein Quantitation and an Introduction to Enzyme Kinetics Flashcards
How do proteins absorb UV light?
Proteins absorb UV light due to the strong absorption by the amino acids tryptophan and tyrosine at approx 280nm.
What is intrinsic protein assays?
Assays which exploit the inherent light absorbing properties of specific amino acids (tryptophan and tyrosine).
What problem do colorimetric protein assays suffer that intrinsic protein assays don’t?
The dye molecules in a colorimetric protein interact with the proteins in a sample, it means the protein has been chemically altered in some way, and thus all colorimetric protein assays suffer from the shortcoming that they are destructive to protein samples.
Types of assays:
a) Biuret assays:
b) Lowry assays:
c) Bradford assays:
a) bind Cu2+ions to proteins making a light blue colour. does not work well with large amounts of proteins but can be used for more modern and sensitive assays. doesnt take too long.
b) similar to biuret assays. it is based on the initial complex of Cu2+ ions and peptide bonds but improved sensitivity comes from the fact that the copper ions reduce a regent called Colin-ciocalteu (FC) which produces a intense purple colour. Is not used regularly but used in modern labs bc of accuracy and consistency. takes some time (1hr)
c) high sensitivity, rapid results, and low cost of reagent.
What are enzymes?
Enzymes are biomolecules (usually proteins), that accelerate the rate of chemical reactions.
What are substrates and products?
The molecules that enzymes act upon are known as their substrates, and the molecules formed by the chemical reaction accelerated by the enzyme are known as products.
enzyme + substrate –> enzyme + product
How can you distinguish between the substrate and product in an assay?
The product and substrate molecules absorb light differently.
What are chromogenic substrates?
Substrates that enzymes can covert into products that can be readily detected by the spectrophotometer.
What happens to the slope of a reaction progress curve based on how the product absorbs light?
If the product absorbs MORE light THAN the substrate, the reaction progress curve will have a positive slope.
If the product absorbs LESS light than the substrate, the curve will have a negative slope.
What is Initial Rate (V0)?
Initial rate, or catalytic rate, is in the first few moments when the enzyme first begins to convert substrate to product.
What does linearity have to do with initial rate?
The initial rate is determined from the reaction progress curve, by determining the slope of the line up until it begins to measurably deviate from linearity.
If we are satisfied that the slope of the reaction progress curve is not changing significantly w/n the error of the instrument, we can be confident that our measured initial rates are close enough to the true initial rates.
Why do enzymes have specificity?
The specificity arises in part because the portion of the enzyme molecule that interacts with the substrate, the active site, has a three-dimensional structure that limits the range of molecules that can fit within in it.
