BCH4160/2011:Notebook/Laura Lee/2011/11/15

Enzymes Kinetics part 2

This project is a continuation of Enzyme Kinetics part 1.

Materials

Refer to Enzyme Kinetics part 1 for materials used.

Procedure

This experiment is a continued project from November 8th, 2011: Enzyme Kinetics part 1.

Baseline Spectrum

Obtain baseline spectrum of 1.2% solution starch and iodine.

1. Dissolve 1mL 1.2% starch, 1mL buffer, and 5μL iodine in a cuvette.
   • Note: Final concentration is 0.6% starch in solution.
2. Measure absorbance with UV spectrophotometer.

Experiment 1

Purpose: Measure starch reaction with cellobiase, without urea or betaine monohydrate.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
3. Repeat experiment for duplicates.

Experiment 2

Purpose: Measure starch reaction with cellobiase with .5 molal concentration of betaine monohydrate.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 0.25mL of betaine monohydrate (for final concentration of 0.5 molal) to cuvette. Shake to mix.
3. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
4. Repeat experiment for duplicates.

Experiment 3

Purpose: Measure starch reaction with cellobiase with 1 molal concentration of betaine monohydrate.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 0.5mL of betaine monohydrate (for final concentration of 1 molal) to cuvette. Shake to mix.
3. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
4. Repeat experiment for duplicates.

Experiment 4

Purpose: Measure starch reaction with cellobiase with 2 molal concentration of betaine monohydrate.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 1mL of betaine monohydrate (for final concentration of 2 molal) to cuvette. Shake to mix.
3. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
4. Repeat experiment for duplicates.

Experiment 5

Purpose: Measure starch reaction with cellobiase with 3 molal concentration of betaine monohydrate.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 1.5mL of betaine monohydrate (for final concentration of 3 molal) to cuvette. Shake to mix.
3. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
4. Repeat experiment for duplicates.

Experiment 6

Purpose: Measure starch reaction with cellobiase with 1 molal concentration of urea.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 0.222mL of urea (for final concentration of 1 molal) to cuvette. Shake to mix.
3. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
4. Repeat experiment for duplicates.

Experiment 7

Purpose: Measure starch reaction with cellobiase with 2 molal concentration of urea.

1. Add 1mL 1.2% starch, 1mL buffer, and 5μL iodine to a cuvette. Shake to mix.
2. Add 0.444mL of urea (for final concentration of 2 molal) to cuvette. Shake to mix.
3. Add 10μL cellobiase solution to cuvette. Quickly mix and place in UV spectrophotomer to measure absorbance.
4. Repeat experiment for duplicates.

Data/Results

Raw data of cellobiase with betaine monohydrate. File:GB experiments raw data.xls

Raw data of cellobiase with urea. File:Urea Experiment Raw Data.xls

Contact

This experiment was done with High Point University students Jigesh Patel and Brittany Webster.