Biochemistry Ldh Assay

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BIOL 3380 Name:_____________________________________

Circle Session: T-PM W-AM W-PM R-AM R-PM F-AM F-PM

Experiment 9 – Pre-lab Homework
Enzyme Kinetics of LDH
This pre-lab homework assignment is due at the beginning of your lab session.

You are provided with the following portion of a protocol: • Determine concentration of enzyme stock solution, if unknown, by taking an A280 nm reading of a 1:100 dilution (in water). Use a total volume of 1 ml in the cuvette.

• Dilute some of the enzyme stock with buffer A to make a 4 mg/ml solution.

• Serially dilute the 4 mg/ml solution with buffer A to make working solutions of 400 µg/ml and 40 µg/ml.

• Prepare 30 µl of each working solution for
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B. Catalysis occurs on a specific site on the enzyme (the active site). The active site is usually less than 5% of the surface area of the protein, and is always in a cleft. The rest of the molecule serves to present the active site in a three dimensional structure that is capable of binding substrate and catalyzing the reaction. Binding to a substrate is very specific, and involves ionic interactions, H bonds and van der Waals forces. C. Only amino acids with polar side chains participate directly in catalysis (Cys, His, Ser, Asp, Glu and Lys). Some reactions require electron acceptors, and since no side chains are good electron acceptors coenzymes and cofactors are required for catalysis of some reactions. D. Enzymes catalyze reactions by several means: ▪ by orienting the substrate molecules. ▪ by increasing the effective concentration of the substrate. ▪ by acid-base catalysis ▪ by covalent catalysis ▪ by using the energy of substrate binding to promote the enzyme catalyzed reaction.

III. Criteria For Establishing The Presence Of Enzymes

Since most of the known biological catalysts are proteins two criteria are generally used for establishing the existence of enzymes. The first is that the rate of a reaction in the presence of an enzyme is greater than the rate in its absence. Because the uncatalyzed rates of most biologically important reactions are effectively zero, the mere

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