You will perform the protocol below for the calf intestinal alkaline phosphatase (CIP) provided. For each reaction, your final enzyme concentration should be 10 nM CIP. Note: Enzymes purchased are typically labelled with their “units of activity” (U), as this relates to how much enzyme is needed to catalyze a reaction. The 100 nM CIP provided has approximately 3 U/mL and was diluted 1 in 1,000 from a 500 U/mL purchased enzyme. 1) Create a table (similar to the one below) to help you determine and keep track of what to add to each of the cuvettes in which your reactions will be measured. The five different concentrations of PNPP should be: 25, 50, 100, 200, 300 μM. Each reaction will be in a final volume of 1 mL and contain 10 nM alkaline phosphatase. Concentrations of stock solutions: 1.0 mM PNPP, 100 nM calf intestinal phosphatase
Enzyme kinetics
In biochemistry, enzymes are proteins that act as biological catalysts. Catalysis is the addition of a catalyst to a chemical reaction to speed up the pace of the reaction. Catalysis can be categorized as either homogeneous or heterogeneous, depending on whether the catalysts are distributed in the same phase as that of the reactants. Enzymes are an essential part of the cell because, without them, many organic processes would slow down and thus will affect the processes that are important for cell survival and sustenance.
Regulation of Enzymes
A substance that acts as a catalyst to regulate the reaction rate in the living organism's metabolic pathways without itself getting altered is an enzyme. Most of the biological reactions and metabolic pathways in the living systems are carried out by enzymes. They are specific for their works and work in particular conditions. It maintains the best possible rate of reaction in the most stable state. The enzymes have distinct properties as they can proceed with the reaction in any direction, their particular binding sites, pH specificity, temperature specificity required in very few amounts.
You will perform the protocol below for the calf intestinal alkaline phosphatase (CIP) provided. For each reaction, your final enzyme concentration should be 10 nM CIP.
Note: Enzymes purchased are typically labelled with their “units of activity” (U), as this relates to how much enzyme is needed to catalyze a reaction. The 100 nM CIP provided has approximately 3 U/mL and was diluted 1 in 1,000 from a 500 U/mL purchased enzyme.
1) Create a table (similar to the one below) to help you determine and keep track of what to add to each of the cuvettes in which your reactions will be measured. The five different concentrations of PNPP should be: 25, 50, 100, 200, 300 μM. Each reaction will be in a final volume of 1 mL and contain 10 nM alkaline phosphatase.
Concentrations of stock solutions: 1.0 mM PNPP, 100 nM calf intestinal phosphatase
![Final [] of
PNPP (μM)
Vol H₂O
(μL)
Vol of 1.0 mM
PNPP (μL)
Vol of 2x
reaction buffer
(μL)
Final [] of
enzyme (nM)
Vol of enzyme
(μL)](/v2/_next/image?url=https%3A%2F%2Fcontent.bartleby.com%2Fqna-images%2Fquestion%2Fa6fdb913-5513-48f0-8c37-c1f95f49bc32%2F3b7500b0-2a6b-4059-bbf8-87d828479317%2Fcgw7mbfj_processed.png&w=3840&q=75)
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can you follow up on how to solve for the volume of water and 2x buffer in depth, then the volume of the enzyme? I don't understand how you got those numbers
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