Enzymatic Assay of Cytochrome c Reductase

Enzymatic Assay of Cytochrome c Reductase Principle & Protocol

1. Introduction

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Enzyme Assay Enzyme assays are laboratory methods for measuring enzymatic activity. They are vital for the study of enzyme kinetics and enzyme inhibition.

Cytochrome c Reductase Cytochrome c reductase is a flavoprotein that completes the oxidation-reduction chain between hexosemonophosphate and cytochrome c.

2. Principle

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Abbreviations: β-NADH = β-Nicotinamide adenine dinucleotide, reduced form β-NAD = β-Nicotinamide adenine dinucleotide, oxidized form Conditions: T = 25 oC, pH = 8.5, A550nm, Light path = 1 cm Methods: Continuous spectrophotometric rate determination

3. Reagents

A B

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300 mM Glycylglycine Buffer, pH 8.5 at 25 oC (Gly Gly)

(Prepare 100 ml in deionized water using Gly-Gly, Hydrochloride. Adjust pH to 8.5 at 25 oC with 1 M NH4OH.)

1% (w/v) Cytochrome c Solution

(Prepare 10 ml in deionized water using Cytochrome c from Horse Heart)

C

7.05 mM β-Nicotinamide Adenine Dinucleotide, Reduced Form (β-NADH)

D

20 mM Potassium Bicarbonate Buffer, pH 8.5 at 25 oC (KHCO3)

E

Cytochrome c Reductase Enzyme Solution (CCR)

(Dissolve the contents of one vial of β-Nicotinamide Adenine Dinucleotide, Reduced Form, Disodium Salt, in 1 ml of Reagent A.)

(Prepare 100 ml in deionized water using Potassium Bicarbonate, Adjust pH 8.5 at 25 oC with 1 M HCl.)

(Immediately before use, prepare a solution containing 0.05 - 0.25 units/ml of Cytochrome c Reductase in cold Reagent D.)

4. Protocol

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4.1 General Procedure

Add reagents

Mix and equilibrate

Record at A550nm

Add reagents

Mix

Record the increase at A550nm

a. Pipette (in milliliters) the following reagents into suitable cuvettes:

b. Mix by inversion and equilibrate to 25 oC. c. Monitor the A550nm until constant, using a suitably thermostatted spectrophotometer. d.Then add: e. Immediately mix by inversion. f. Record the increase in A550nm for approximately 5 minutes. g. Obtain the ∆ A550nm / minute using the maximum linear rate for both the Test and Blank.

Linear regression

4. Protocol

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4.2 Calculations

21.0 = ∆ millimolar extinction coefficient between oxidized and reduced cytochrome c at pH 8.5 RM = Reaction Mix Unit Definition: One unit will reduce 1.0 μmole of oxidized cytochrome c per minute at pH 8.5 and 25 oC. Final Assay Concentraton: In a 3 ml reaction mix, the final concentrations are 30 mM glycylglycine, 0.033% cytochrome c, 0.24 mM ß-NADH and 0.005 - 0.025 units of cytochrome c reductase

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