Lab 3 - Use of Enzymes as Reagents; Coupled Enzyme Assays...

Info iconThis preview shows pages 1–4. Sign up to view the full content.

View Full Document Right Arrow Icon
Use of Enzymes as Reagents; Coupled Enzyme Assays and Enzyme Kinetics 1
Background image of page 1

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Use of Enzymes as Reagents; Coupled Enzyme Assays and Enzyme Kinetics University of Texas at Austin Department of Chemistry and Biochemistry Introduction A coupled enzyme assay is used when determing a substrate or product concentration, to monitor the reaction of a coenzyme, and for more complex systems that are less stable to biochemically quantify. We are using the couple enzyme assay to determine the amount of glucose in a sample. This involves the reaction of hexokinase, an enzyme, with glucose, which then is a substrate for a second enzyme glucose-6-phosphate dehydrogenase (G6PD). G6PD then reduces NAD + to NADH, which absorbs at 340 nm. 12,13 We are also using the couple enzyme assay to determine the amount of ethanol in a sample. This is primarily used in drug testing and law enforcement and involves the oxidation of ethanol to acetaldehyde. The buffer used contains semicarbazide, which irreversibly binds acetaldehyde so that NAD + can’t be formed again, which would give us an inaccurate result. 12,13 In the second part of the lab, we are using a phosphatase to hydrolyze p- nitrophenol phosphate. We will then be using the Michaelis-Menton equation to help us create a Lineweaver – Burk plot, which is a great way to analysis enzyme kinetics. 14 ] [ ] [ max 0 S K S V V m + = (1) ) 1 ] [ 1 )( ( ) / 1 ( max max 0 V S V K V m + = (2) Data and Observations When we carried out our experiments, none of the procedures deviated from the lab manual’s procedures. Part 1: We obtained an absorbance of a glucose standard solution (10mM), glucose reagent, and water Tube Number A 340 Glucose Conc. (mM) 1 2 0.911 0.0971 3 1.010 0.194 4 1.019 0.243 5 1.052 0.291 Table 1 . Absorbance at 340 nm of a glucose assay 2
Background image of page 2
We obtained an absorbance of an unknown glucose solution, glucose reagent, and water Tube Number A 340 2 0.877 Table 2 . Absorbance at 340 nm of an unknown glucose assay We obtained an absorbance of an ethanol standard solution (50mM),
Background image of page 3

Info iconThis preview has intentionally blurred sections. Sign up to view the full version.

View Full DocumentRight Arrow Icon
Image of page 4
This is the end of the preview. Sign up to access the rest of the document.

This note was uploaded on 09/07/2009 for the course CH N/a taught by Professor Genemcdonald during the Spring '09 term at University of Texas at Austin.

Page1 / 10

Lab 3 - Use of Enzymes as Reagents; Coupled Enzyme Assays...

This preview shows document pages 1 - 4. Sign up to view the full document.

View Full Document Right Arrow Icon
Ask a homework question - tutors are online