Introduction to Data Gathering and Analysis Using Enzyme Kinetics Data as Experimental Models

Foundations of a scientific study is the careful collection, organization and analysis of data from which conclusions are formed. Scientific laws and theories are established through such experimentation, data gathering and correct analysis of generated data Data gathering and analysis are important skills we should develop

Gathering data is a frequent part of solving problems and satisfying curiosity. When we look up information to answer a question or to formulate new questions, we are gathering and analyzing data. When we conduct surveys and draw conclusions from them, we are gathering and analyzing data.

The analysis includes graphs as well as numerous complex mathematical tools.

We use statistics to provide meaning to what otherwise would be a collection of numbers and/or values

It is the study of the chemical reactions that are catalyzed by enzymes. In enzyme kinetics the reaction rate is measured and the effects of varying the conditions of the reaction investigated. Studying an enzyme's kinetics in this way can reveal the catalytic mechanism of this enzyme, its role in metabolism and how its activity is controlled.

Collected data are organized in the form of tables and represented by graphs for analysis Graphs provide us the trends of the data. It will show scientific data on a timed basis using enzyme kinetics. Basic statistics will be reviewed for data analysis

Perform the proper skills of data gathering and analysis. Construct appropriate tables and figures for gathered data. Explain the importance of statistical analysis in the gathered data. Analyze the enzyme kinetics data using appropriate statistical tool.

Preparation of Catalase Extract

 10g of liver with 100ml of PB was homogenized

for 30 secs.  Homogenate was filtered with filter paper  Diluted to final conc. of 8g per buffer liter  Solution labeled to be Catalase Enzyme Stock

CATALASE ENZYME STOCK !!!

Preparation of Hydrogen Peroxide Substrate

 0.2M of H2O2 was diluted with PB to the ff. conc.

0.005 0.010 0.015 0.020 0.025

0.050 0.075 0.100 0.150 0.200

20 ml of final volume should be prepared for these dilutions

Yeah! H2O2

H2O2 (M) 0.005 0.010 0.015 0.020 0.025 0.050 0.075 0.100 0.150 0.200

H2O2 (ml) 0.5 1 1.5 2 2.5 5 7.5 10 15 20

K4PO4 (ml) 19.5 19 18.5 18 17.5 15 12.5 10 5 0

Catalase Assay
 Ten 50 ml beakers prepared with their appropriate    

H2O2 conc. Beakers put to equilibrate for 10 mins. Place filter paper in the beakers Observe its when the disc hits the bottom of the beaker. Perform it in three replicates Determine and record.

Table 3.1 Catalase Reaction on Different Substrate Concentration in Relation to Time and Velocity Substrate Concentration (M) 0.0005 0.010 0.015 0.020 0.025 0.050 0.075 0.100 0.150 0.200 1296 127.67 123.33 30.90 33.18 12.97 6.35 10.24 5.43 4.09 Time (s) Velocity (cm/s) 0.00046 0.01 0.012 0.05 0.05 0.12 0.24 0.15 0.30 0.37

Table 3.1 Catalase Reaction on Different Substrate Concentration in Relation to Time and Velocity Substrate Concentration (M) 0.0005 0.010 0.015 0.020 0.025 0.050 0.075 0.100 0.150 0.200 1793 236 113 60 32.71 20.12 11.82 8.22 3.34 2.19 Time (s) Velocity (cm/s) 0.000948 0.007 0.02 0.03 0.05 0.08 0.14 0.21 0.51 0.78

Figure 3.1 Reaction Velocity versus Substrate Concentration in an Enzyme-Catalyzed Reaction

Figure 3.1 Reaction Velocity versus Substrate Concentration in an Enzyme-Catalyzed Reaction

The reaction rate also increases in proportion to substrate concentration, but only to a certain point, where it reaches a maximum velocity as shown in the Figures above. The maximum velocity is proportional to the amount of enzyme present.

The hyperbola depicts 3 observations that caused the curve. At low (1), velocity is directly proportional to substrate. At intermediate (2), velocity is depended on substrate. And at high (3), velocity is independent of substrate.

At high, saturation has been achieved. This is the property of Enzyme-Catalyzed reactions, wherein it cannot increase the reaction velocity beyond a finite upper value even with the increasing of substrate concentrations.

1/substrate 200 100 66.67 50 40 20 13.33 10 6.62 5

1/velocity 2173.91 100 83.33 20 20 8.33 4.17 6.67 3.36 2.70

1/substrate 200 100 66.67 50 40 20 13.33 10 6.62 5

1/velocity 1054.85 142.86 50 33.33 20 12.5 7.14 4.76 1.96 1.28

Reciprocal Data of Group 3& 4

Reciprocal Data of Group 1 & 2

Figure 3.2 Plot of the Rate of Reaction versus Substrate Concentration in an Enzyme-Catalyzed Reaction.

Figure 3.2 Plot of the Rate of Reaction versus Substrate Concentration in an Enzyme-Catalyzed Reaction.

The figures above, indicates the R value to be almost 98% of the variation in 1/v (y) is due to the variation in 1/s (x). In addition, if we take the square root of R we can determine that the correlation ,r, is almost 1. The r being almost 1 indicates an excellent fit between the data points and the regression line. This then summarizes as 1/s increases, 1/v increases.

Regression analysis is applied as our statistical tool since we have an independent and a dependent variable. Our independent variable is the molarity of H2O2 and dependent to be the mean time. This shows that time varied due to the fixed concentrations of our H2O2.

Group 3 and 4
 Kmax = 0.5 / vmax = 0.37 / 2

= 0. 18 therefore Kmax is 0.063M

Group 1 and 2
 Kmax = 0.5 / vmax = 0.78 / 2

= 0.39 therefore Kmax is 0.1395M

The data of groups 1 and 2 has a high Km compared to the data of groups 4 and 5. High Km value means a high saturation point of the enzyme which results from a low affinity of the enzyme for the substrate.