Enzyme Kinetic report - effect of substrate concentration on PDF

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effect of substrate concentration on reaction velocity of alkaline phosphatase...

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100156554

Alex Martin

Effect of substrate concentration on the reaction velocity of alkaline phosphatase

Intro: Enzymes are the largest group of catalytic proteins; they are organic catalysts that increase the rate of a reaction that would be otherwise slow or imperceptible to occur with the absence to a net change in their structure (Dixon 1953). An Enzyme will allow reactions to occur by providing an alternative biochemical pathway in turn lowering the activation energy for a reaction (Klug et al 2014). Alkaline Phosphatase is an enzyme found in many organisms from a human body to bacteria which helps break down proteins as it is a hydrolase (Berger et al 1988). It has many origins and so varies as to what subject it will be breaking down, such as a pregnant woman it is made in the placenta but the majority is synthesized in the liver in humans (millán 2006). Alkaline Phosphatase is responsible for removing phosphate groups from many molecules; proteins, nucleotides and alkaloids, this process is called dephosphorylation (Hoylaerts et al 2015). The aim of the experiment was to determine the maximum velocity of the enzyme reaction by changing the substrate concentration to calculate the two parameters of Vmax and Km. Thus consequently showing the effect of the substrate concentration on the reactions velocity. Method: A sample of six different solutions were made into cuvettes which the enzyme was added too. A spectrophotometer was used to make this experiment possible and measured the absorbency at 405 nm. Every 15 seconds a reading was taken and recorded for each concentration (0.5, 0.8, 1.0, 2.0, 5.0, 10.0 (mM))

100156554

Alex Martin

Results: The absorbance was measured at different concentrations (0.5 mM, 0.8 mM, 1.0 mM, 2.0 mM, 5.0 mM, 10 mM) with 15 sec intervals using a spectrophotometer (405 nm). Table 1. The absorbance values below are shown for each of the concentrations with all 15 sec intervals (15-120 sec). Absorbance (405 nm) Concentration of p-nitrophenyl phosphate Time (sec)

0.5 mM

0.8 mM

1.0 mM

2.0 mM

5.0 mM

10.0 mM

15

0.024

0.081

0.067

0.134

0.241

0.308

30

0.043

0.089

0.080

0.169

0.340

0.419

45

0.061

0.097

0.093

0.201

0.437

0.529

60

0.078

0.105

0.105

0.232

0.529

0.641

75

0.095

0.113

0.116

0.262

0.614

0.752

90

0.110

0.120

0.129

0.288

0.636

0.859

105

0.125

0.126

0.140

0.312

0.781

0.962

120 0.139 0.133 0.151 0.338 0.863 1.053 This Data in table 1 was plotted on a graph (Figure 1) shown in Appendix 1. A class data set was collated with the reaction velocity calculated for each concentration. To calculate the reaction velocity, the last minus the first absorbency was calculated for each concentration. Each was divided by 105 (the difference in time) to calculate per second and then times by 60 to get the velocity of reaction to be per min.

100156554

Alex Martin

Table 2. shows the concentration of p-nitrophenyl phosphate (mM) with the corresponding velocity of reaction from the class data set respectively. Concentration (mM) of pnitrophenyl phosphate (S)

0.5 0.8

1

Velocity of reaction (absorbance change/min) (V) 0.085

0.123 0.147

2

0.233

5

0.349

10

0.433

The data was then inverted to 1 over ‘X’ to make it per min for both data sets. Table 3. the data shown in the new inverted form and a change in units shown in the corresponding columns. 1/S (mM-1)

1/V (absorbance change/min-1)

2

11.765

1.25

8.130

1

6.803

0.5

4.292

100156554

Alex Martin

0.2

2.865

0.1

2.309

A Lineweaver-Burk graph was constructed to calculate Vmax and Km values and a single straight line from the data.

14

12

1/Velocity (absorbance change/ min-1)

f(x) = 4.98 x + 1.84 10

8

6

4

2

0

0

0.5

1

1.5

2

2.5

1/[Substrate] (mM-1)

Figure 2 A Lineweaver-burk graph showing the equation of the line, the points plotted and the line being extrapolated onto the negative ‘X’ axis to aid finding Km and Vmax. Using simple algebra rearranging the equation of the line with Y=0 then X=0 the X and Y intercepts were calculated. For the Y intercept; X=0 so therefore Y=1.8361 (4.9797 x 0 = 0). For the X intercept; Y=0 so therefore 0= 4.9797x + 1.8361… -1.8361= 4.9797x (shift to other side) … -1.8361/4.9797=x.

100156554

Alex Martin

Table 4 the line intercepts calculated for both X and Y are shown below. Value at the intercept of the X-axis (1/S)

-0.369

Value at the intercept of the Y-axis (1/V)

1.836

The following calculations were used to determine Km: -1/Km = -.0369, therefore, Km =2.710 mM. The following calculations were carried out to determine the value of V max: 1/Vmax = 1.836, therefore, Vmax = 0.545 absorbance change min-2. Using the Beer Lambert Law (A=cl), the units for Vmax were changed to µmol min-1: 0.545/1 X (1.8 x 104) = 3.027 x 10-5 M 3.027 x 10-5 X 0.003 = 9.083 x 10-8 mols 9.083 x 10-8 X 106 = 9.083 x 10-2 µmol min-1 Vmax describes the maximum velocity and is proportional to the amount of enzyme added to the assay system. Table 5 Values of Km and Vmax after calculation Km value (mM)

2.710

Vmax (μmol min-1)

0.545

100156554

Alex Martin

Discussion: The Vmax value is relatively low meaning that the enzyme does not convert much substrate to product per unit of time when the enzyme is saturated with substrate. Thus meaning the reaction was slow and has lower levels of final output comparatively, (Hoylaerts et al 2015) this could down to many factors that could influence the rate. One factor that could affect the reaction Is temperature; the cuvettes were made up and left on the lab bench until needed so they had time to adapt to room temperature but also it is unknown where they were stored prior whether they were at optimum temperature etc. enzymes work best around 37.5 C (Shimoboji et al 2003) and so forth would influence the reaction rates present. With the enzyme Alkaline Phosphatase, the more product that is produced the more the solution absorbs in the spectrophotometer so therefore the results can be continuous, meaning that a graph can be drawn over time to be converted and gradual changes can be seen and mapped due to the intermediate time intervals. This lets a full picture be seen and is comparable to other experiments (Millán J.S 2006). A possible error in the experiment is that what part of the solution was measured by the spectrophotometer, if the solution was shaken or mixed in anyway will off needed to be consistent (Hasani-Tabatabaei et al 2009). Still this will not give a true reading as the solution will always be moving, to combat this a set routine could be set and also repeat readings of each solution will make the results more exact. The Km value was 2.710 thus meaning the larger Km the lower the affinity of the substrate for the enzyme. So forth meaning it takes a greater concentration of the substrate for the enzyme to be half saturated in comparison to a lower K m value. The reaction therefor was not as spontaneous as another and was slower with a lower maximum velocity (Schnell and maini 2003). The effect of substrate concentration on the reaction velocity is proven in Figure 1 as the higher absorbance is clearly shown over time with a much higher absorption rate. This is absorption is possible due to the product p-nitrophenol having a strong absorbance.

100156554

Alex Martin

References: Berger J, Hauber J, Hauber R, Geiger R, Cullen B.R (1988) Secreted placental alkaline phosphatase: a powerful new quantitative indicator of gene expression in eukaryotic cell, Roche Institute of Molecular Biology, Gene, 66(1), 1-10. Dixon M (1953) The effect of pH on the affinities of enzymes for substrates and inhibitors, Biochemical Laboratory, University of Cambridge, BioChem J, 55(1), 161170. Hasani-Tabatabaei M, Yassini E, Moradian S, Elmamooz N (2009) Color stability of dental composite materials after exposure to staining solutions: a spectrophotometer analysis, Operative Dentistry Department, Faculty of Dentistry, Tehran University/Medical Sciences, Tehran, Iran, journal of Islamic dental association of Iran, 21(1), 69-78. Hoylaerts M.F, Kerckhoven S.V, Kiffer-moreira T, Sheen C, Narisawa S, Millán J.L (2015) Functional significance of Calcium binding to Tissue-nonspecific Alkaline Phosphatase, Department of Cardiovascular sciences, Center for Molecular and Vascular Biology, University of Leuven, Belgium, ONE, 10(3), 56-60. Klug W.S, Cummings M.R, Spencer C.A, Palladino M.A (2014) Concepts of genetics, San Francisco: Benjamin-Cummings publishing company, 10th, 598-604. Millán J.S (2006) Alkaline Phosphatases; Structure, substrate specificity and functional relatedness to other members of a large superfamily of enzymes, Burnham Institute for Medical Research, Purinergic Signal, 2(2), 335-341. Schnell S, Maini P.K (2003) A century of enzyme kinetics. Should we believe in the Km and vmax estimates?, Comments in Theoretical Biology, 8 (2-3), 169-187. Shimoboji T, Larenas E, Fowler T, Hoffman A.S, Stayton P.S (2003) TemperatureInduced Switching of Enzyme Activity with Smart Polymer−Enzyme Conjugates, Department of Bioengineering, University of Washington, Seattle, Washington, Bioconjugate, 14(3), 517-525.

100156554

Alex Martin

Appendix: Figure 1; A graph to show the absorbency changes over time at six different concentrations....