Essay On Reaction

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This lab experiment is aimed at studying the rate of reaction involving enzymes by observing the change in absorbance when the reaction is carried out. This experiment will illustrate both the methods of using the linked assay method.

Introduction

The reaction rate of reactions involving enzymes is studied spectrophotometrically by monitoring the change in absorbance at different intervals during the reaction. The accurate measurement of glucose is important in the diagnosis and management of hyperglycemia and hypoglycemia. The enzymes that will be used in this experiment are Hexokinase (HK) and Glucose-6-Phosphate Dehydrogenase (G6PDH). The nicotinamide coenzymes are Nicotinamide Adenine Dinucleotide (NAD) and Nicotinamide Adenine Dinucleotide Phosphate (NADP). These two are collectively also known as NAD(P). The oxidized forms are NAD(P)+ and the reduced forms are NAD(P)H. The reduced forms have an absorbance peak at 340 nm which is not present in the oxidized forms. Hence, the increase or decrease in absorbance signifies the reduction or oxidation.
This method is also used to measure the rate of reactions of enzymes which do not involve NAD(P) as coenzyme, but it may be coupled to another enzyme reaction that does.
The same method is also used to determine the concentrations of certain metabolic intermediates by metabolizing them with an enzyme that either uses NAD(P) itself or produces a product which then reacts with an NAD(P) linked enzyme. The metabolism of the test substrate is linked stoichiometrically to the oxidation or reduction of NAD(P) and its concentration is determined by the measurement of the change in coenzyme concentration.
The molar extinction coefficient of NAD(P)H at 340 nm is taken as 6.22 x 103 because it is not feasible to determine the value of the coefficient practically in this experiment due to the oxidation tendency of NAD(P)H during storage which makes it difficult to retain a standard curve.

The enzyme Hexokinase catalyzes the reaction between Glucose and Adenosine TriPhosphate to form Glucose-6-Phosphate (G6P) and Adenosine DiPhosphate (ADP). In the presence of Nicotinamide Adenine Dinucleotide (NAD), G6P is oxidized by the enzyme Glucose-6-Phosphate Dehydrogenase to 6-Phosphogluconolactone and reduced Nicotinamide Adenine Dinucleotide (NADH). The consequent increase in NADH concentration is directly proportional to the glucose concentration and can be measured spectrophotometrically at 340 nm.

The reaction through which the experiment reaches completion is:

It is a coupled chemical reaction. In the first reaction, Hexokinase catalyzes the phosphorylation of glucose by ATP producing ADP and Glucose-6-Phosphate. In the second reaction, Glucose-6-Phosphate is oxidized by Glucose-6-Phosphate Dehydrogenase to 6-Phosphogluconolactone with the reduction of NAD+ to NADH. The stepwise reactions are:
Glucose + ATP → Glucose 6-Phosphate + ADP
Glucose-6-Phosphate + NAD+ → 6-Phosphogluconolactone + NADH + H+

Materials

The materials that will be needed to complete the experiment are:
Low Volume Plastic Cuvette
Pipettes of suitable volume
Spectrophotometer
Deuterium Lamp
Reagents
The following reagents will be needed during the experiment:
Buffer
Adenosine TriPhosphate (ATP)
Nicotinamide Adenine Dinucleotide Phosphate (NADP)
Glucose
Glucode-6-Phosphate Dehydrogenase (G6PDH)
Hexokinase (HK)
Two Glucose Solutions (labeled A & B)
Precautions
Proper safety and experiment related precautions should be taken and appropriate protective equipments should be employed. Wear lab coat, gloves and safety goggles. Place all equipments that contact the specimen in a biohazard waste container which must be disposed off properly. Discard all glassware into sharps waste container. Protect all the surfaces that may come into contact with the specimen using a disposable absorbent paper which should be discarded into the biohazard waste container after the experiment. Wipe all work surfaces with an appropriate material. Protect the specimen from any foreign bodies, for those may alter the results completely. Also refer to Material Safety Data Sheets (MSDSs) for the respective precautions of the reagents.

Procedure

Measurement of the Hexokinase activity
The significant parameter that needs to be observed during the reaction is the rate of the reaction. The reaction rate is obtained by recording different values of absorbance at 340 nm and plotting it against time. The slope of the curve denotes the rate of the reaction.

Proceed with the experiment with the steps mentioned below:

Pipette the following amounts of solutions into the cuvette and mix by inversion:
Buffer 0.6 mL
ATP 0.1 mL
Glucose 0.1 mL
G6PDH 0.02 mL
Zero the spectrophotometer.
Start the reaction by adding 0.02 mL of 1/500 strength Hexokinase and immediately mix well by inversion.

Record the absorbance at 340 nm for about 3 minutes at an interval of 15 seconds each.

Plot absorbance versus time.
The initial slope of the plot is the required rate of reaction.
Repeat the procedure, this time using 0.01 mL of Hexokinase.

Measurement of Glucose Concentration

Glucose is measure by the same method but this time, excess Hexokinase solution should be used. In this case, the final NADPH concentration at the completion of the reaction is the significant parameter to be noticed instead of the rate of the reaction. Ideally, the reaction should be fast due to the use of the stronger solution of Hexokinase of strength 1/100.

Proceed with the experiment with the steps mentioned below:

Pipette the following amounts of solutions into the cuvette:
Buffer 0.6 mL
ATP 0.1 mL
G6PDH 0.02 mL
HK of strength 1/100 0.02 mL
Zero the spectrophotometer.
Start the reaction by adding 0.1 mL of A or B Glucose solution.

Record the absorbance at completion.

If the value is greater than 1, repeat the procedure using a diluted solution of A or B.
Calculate the concentration of Glucose in the unknown samples using the extinction coefficient of NADPH.
Interpretation of Results
While measuring the Hexokinase activity, plot the recorded values of absorbance at 340 nm versus time and measure or calculate the slope of the plot. The initial slope is the required rate of reaction.
Slope= x2-x1y2-y1=0.231-0.0560-15=0.004022
So, the HK activity is 0.004022 nm/s or 0.24132 nm/min.
While measuring the Glucose concentration, record the absorbance at only the completion point of the reaction. Utilize the value which is less than 1 and calculate the concentration of the unknown samples of Glucose using the extinction coefficient of NADPH. The value so calculated is the desired Glucose concentration.

The Glucose concentration is calculated by the following formula:

mg glucoseml= ∆ATVGlucose Molecular Weight(F)ESV(Conversion factor for µg to mg)
Where ∆A = Atest - Atotal blank
Atotal blank = Asample blank + Areagent blank
TV = Total Assay volume (ml)
SV = Sample volume (ml)
Glucose MW = 180.2 µg/µmoles
F = Dilution Factor
ℰ = Millimolar Extinction Coefficient for NADH at 340 nm = 6.22

Conversion factor for µg to mg

mg glucoseml= 1.163-0.664-0.3860.95180.20.016.220.11000
mg glucoseml=3.11×10-4
The time courses of the reactions with various amounts of glucose added as substrate are shown in Figure 1. There was a little difference between the rates of reactions with 20 µl, 40 µl and 60 µl glucose indicating that these amounts of substrate were just sufficient not to limit the reaction.
Figure 1
The present study shows that the reference method for the determination of glucose, the Hexokinase/glucose-6-phosphate Dehydrogenase method, can be carried out kinetically over a wide range of glucose concentration if appropriate assay conditions are used.
The usual values for human serum or plasma lie within the range of 3.89 – 5.83 mmol/L (7 – 10.5 mg/ml) and that of urine lies within the range of 0.28 – 0.83 mmol/L (0.5 – 1.5 mg/ml).
The values obtained experimentally for Hexokinase activity and glucose concentration lie within the normal range and the Hexokinase activity complements the glucose concentration.