experiment 3 kinetics

Title: Experiment 3: Kinetic studies with alkaline phosphataseObjective(s):1. To prepare the standard curve for p nitrophenol2. To study the effect of the addition of Mg2+ ions an alkaline phosphateIntroduction:Phosphatases are enzymes that catalyze the hydrolysis of esters of phosphoric acid. They occur in the cells and extracellular fluids of a wide range of organisms. This large and complex group of enzymes falls into four general types based on the chemical nature of the substrate or the type of hydrolytic reaction that is catalyzed .This large and complex group of enzymes falls into four general types based on the chemical nature of the substrate or the type of hydrolytic reaction that is catalyzed.

One group, the phosphomonoesterases, hydrolyzes monoesters of phosphoric acid such as -glycerophosphate or glucose 6-phosphate. Some phosphomonoesterases are highly substrate-specific. For example, in gluconeogenesis, fructose-1, 6-bisphosphatase specifically converts fructose 1,6-bisphosphate to fructose 6-phosphate and inorganic phosphate. Other phosphomonoesterases react with a broad range of substrates, which share common structural motifs. The phosphomonoesterases that lack substrate specificity are classified as acid or alkaline phosphatases based on their pH optima. Acid phosphatases function best at around pH 5.0 and are inhibited by fluoride ion but not by divalent cation-chelating agents. The alkaline phosphatases have pH optima of about 9.0 and are not generally sensitive to fluoride ion but are inhibited by divalent cation-chelating agents like EDTA (ethylene diamine tetraacetic acid, disodium salt).Low level of alkaline phosphatase can cause, Hypophosphatasia. Hypophosphatasia is a genetic metabolic bone disease that is quite rare in occurrence but fatal to the sufferer. Some of the identifiable symptoms are skeletal hypomineralization, mild respiratory problems, progressive osteomalacia, etc. The patients of hypophosphatasia have very low alkaline phosphatase levels in their blood serum. Such patients often lose their primary teeth much before the standard age. Other than that it can also cause aolastic anemia or chronic Myelogenous Leukemia. In this experiment, the alkaline phosphatases kinetic studies are carried out on the effect of pH, inhibitors and divalent cation. At the beginning of the experiment the standard curve is drawn against the amount of p-nitrophenol. The p-nitrophenol amount used in the assay is determined from the formula of; (Amount of 0.3mM p-nitrophenol) x 10-3L X (0.3 X 10-3mol/L) This graph is used as standard to determine the amount of p-nitrophenol have been consumed in the pH, inhibitor and divalent cations reaction. The alkaline phosphatases have binding sites for Zn2+ and Mg2+, on which enzyme activity is dependent. In this experiment, the effects of addition of Mg2+ ions on alkaline phosphatase are carried out. Next, with variable pH, beginning from pH 7.0, pH 7.5, pH 8.0, pH 8.5 and pH 9.0. Varying pH levels may have a direct effect on the alkaline phosphatase due to the presence of ionizable residues in the catalytic site of the enzyme.

Materials:

Bovine or calf intestinal alkaline phosphatase (Sigma), diluted 1:10,000 with 50 mM Tris-HCl, pH 8.0 buffer, containing 1 mg/ml bovine serum albumin, water bath, p-nitrophenol phosphate in buffer (2.70 mM), p-nitrophenol in buffer (0.3 mM), monobasic sodium phosphate, L- phenylalanine, 50 mM Tris-HCl, pH 7.0, 7.5, 8.0, 8.5, 9.

Procedure:

A) Preparation of standard curve for p-nitrophenolTube123456

0.3nM p-nitrophenol ( ml)-0.050.10.20.40.6

Distilled water(ml)3.02.952.902.802.602.40

1. Following tubes were prepared2. Absorbance was read at 400 nm using tube 1 as a blank3. The amount in mol of p-nitrophenol for each tube4. A graph has been plotted ( absorbance against amount of p-nitrophenol in umol)B) Effects of divalent cationTube 1 1234567

2.70 mM substrate( ml)0.60.60.60.60.60.60.6

50mm tris-HCl pH 8.02.42.32.22.01.81.61.4

15nM MgCl2-0.10.20.40.60.81.0

1. The following tubes were prepared2. Each of the above reaction mixture is added to a suitable cuvette and the reaction was started by adding 20 ul of an enzyme.3. Was mixed by invert4. The cuvette was placed in the spectrophotometer5. Absorbance was read for every 30 s for 3 min at 400 nm6. The A/min was determined.Agraph was plotted in (umol/min) against the MgCl2 concentration

C) Effect of the pH

1. The tubes were prepared same as in B table but the buffer was substituted with buffer with different pH which are 7.0,7.5,8.0,8.5,9.02. A graph of activity against pH was plotted.

Results:

(A) Preparation of standard curve for p-nitrophenol

Tube123456

0.3mM p-nitrophenol (ml)-0.050.10.20.40.6

Distilled water (ml)32.952.92.82.62.4

[p-nitrophenol] (M)-0.0150.0300.0600.1200.18

Absorbance at 400nm00.0180.0410.0560.0720.136

mM convert to M Tube 2:0.3m 0.00005 L = 1.5 10-5 1000 mol = 0.015 molTube 3: 0.3m 0.0001 L = 3 10-5 1000 mol = 0.030 molTube 4:0.3m 0.0002 L = 6 10-5 1000 mol = 0.060 molTube5:0.3m 0.0004 L = 1.2 10-4 1000 mol = 0.120 mol

Tube6: 0.3m 0.0006 L = 1.8 10-4 1000 mol = 0.180 mol

Figure 1 Graph of absorbance againt amount of p-nitrophenol

(B) Effect of divalent cations

TubeTime(min)Absorbance at 400nm

1234567

00.0330.0300.0390.0500.0320.0450.055

0.50.0350.0350.0480.0540.0360.0500.059

1.00.0390.0380.0520.0580.0410.0550.065

1.50.0410.0420.0560.0630.0470.0600.071

2.00.0450.0460.0610.0680.0520.0660.076

2.50.0480.0490.0650.0730.0590.0710.082

3.00.0510.0530.0700.0780.0640.0770.088

Table 1: Absorbance vs Time (min)

Calculation of p-nitrophenol

Absorbance = slope (m) concentrationY = mx, Y is absorbance, m is slope, x is concentrationSlope from standard curve from part A, so m = 0.7371In order to find concentration, use the absorbance / slope

Example:Tube 1 with Time = 0:Concentration = = 0.045 mol Time = 0.5 minConcentration = = 0.047 mol Time = 1 minConcentration = = 0.053 molTime = 1.5 min Concentration = = 0.056 mol

TubeTime (min)[p-nitrophenol] (mol)

1234567

00.0450.0410.0530.0680.0430.0610.075

0.50.0470.0470.0650.0730.0490.0680.080

1.00.0530.0520.0710.0790.0560.0750.088

1.50.0560.0570.0760.0850.0640.0810.096

2.00.0610.0620.0830.0920.0710.0900.103

2.50.0650.0660.0880.0990.0800.0960.111

3.00.0690.0720.0950.1100.0870.1040.120

Table 2: [p-nitrophenol] (mol) vs Time (min)

Graph 1: Tube 1 [p-nitrophenol] (mol) vs Time (min)


Graph 3: Tube 3[p-nitrophenol] (mol) vs Time (min)





Calculation of MgCl2 concentration (M) for the graph of activity (mol/min) vs MgCl2 concentration (M)

Tube1234567

15 mM MgCl2 (mL)-0.10.20.40.60.81.0

MgCl2 concentration (M)-1.53.06.09.012.015.0

Table 3. Conversion of MgCl2 concentration from mL to M

Conversion of the concentration from mM was converted to M by:Tube 215 m x 1x10-4 L x 1000 mol = 1.5 MTube 315 m x 2x10-4 L x 1000 mol = 3.0 MTube 415 m x 4x10-4 L x 1000 mol = 6.0 MTube 515 m x 6x10-4 L x 1000 mol = 9.0 MTube 615 m x 8x10-4 L x 1000 mol = 12.0 MTube 715 m x 1x10-3 L x 1000 mol = 15.0 M

MgCl2 concentration (M)Enzymatic activity (mol/min)

1.50.0101

30.0131

60.0136

90.0149

120.0143

150.0151

Table 4.MgCl2 concentration (M) vs Enzymatic Activity (ol/min).

Graph 8. Enzymatic Activity (mol/min) vsof Magnesium Chloride concentration (M)

C) Effect of pH on the reaction

Tubes with Absorbance Value

Time (minute)12345

00.7490.5120.6930.8980.900

0.50.7550.5131.0130.7980.899

1.00.7600.5151.0240.7920.910

1.50.7650.5161.0270.7950.922

2.00.7730.5191.0300.8010.936

2.50.7790.5221.0360.8090.950

3.00.7850.5261.0420.8130.964

Table 1. Absorbance recorded in 30 seconds time interval with different pH of Tris-HClCalculation of Tris-HCl concentrationConcentration = Slope, m = 0.7371Example:Tube 1, time = 0 minuteConcentration of Tris-HCl = = 1.016 mol

TubeTime (min) Concentration of Tris-HCl at different pH (mol)

1 (pH 7)2 (pH 7.5)3 (pH 8)4 (pH 8.5)5 (pH 9.0)

01.0160.6950.9401.2181.221

0.51.0240.6961.3741.0831.220

1.01.0310.6991.3891.0741.235

1.51.0380.7001.3931.0791.251

2.01.0490.7041.3971.0871.270

2.51.0570.7081.4061.0981.289

3.01.0650.7141.4141.1031.308

Table 2. Concentration of Tris-HCl at 30 seconds time interval with different pH

Tris-HCl pHEnzyme activity (mol/min)

7.00.0165

7.50.0061

8.00.1067

8.5-0.0216

9.00.0310

Table of enzyme activity vs pH of the buffer

Discussion

A standard curve is just a plot of two different parameters and the curve reveals the relationship between the two parameters. Under alkaline conditions, the p-nitrophenolate anion absorbs light at 400-450 nm. The amount of enzyme present is therefore determined by measuring the amount of p-nitrophenolate anion produced in the reaction. However to make this estimation a standard curve must be prepared so that the amount of yellow-orange colour can be translated into the amount of p-nitrophenol produced.

The equation for this reaction is p-nitrophenyl phosphate + H2O p-nitrophenol + H3PO4

As shown by the graph in result section A , it indicates that the rate of absorbances is linearly proportional to against amount of p-nitrophenol in mol. And the equation for this graph is Y = 0.7321X.

Enzymes are proteins that act as biological catalysts that are either essential for a reaction to occur or may increase the speed of the reaction. Some enzyme require the presence of either a cofactor or a coenzyme to catalyse the reaction. (Brennan, n.d.)The specific function of the cofactor may vary according to the enzyme. Each enzyme has its respective sequence of reaction steps which the cofactor play a role in (Brennan, n.d.). Cofactors are required by the enzymes to facilitate the electrons transfer needed in the formation and breaking of bonds in the reaction mechanism. (JAKUBOWSKI, 2014) Divalent cations are cations that have a charge of +2. Magnesium ion, Mg2+ is a divalent cation. It is a cofactor to alkaline phosphathase. A cofactor is a non-protein molecule or ion required by the enzyme when underdoing its enzyme activity. Throughout the experiment the concentration of enzyme, which is alkaline phosphotase, and of the concentration of the cofactor in the form of magnesium chloride, MgCl2. Tube 1 acted as a control to observe the enzymatic activity of phosphotase without the cofactor. Based on Graph 2, the lowest among all the tubes. This indicates that the phosphotase is capable of hydrolyzing p-nitrophenyl phosphate to p-nitrophenol without the presence of Mg2+. However, the presence of the cofactor does yield a much higher enzyme activity.

The concentration of MgCl2 increases from Tube 2 7. This will help in identifying the optimal concentration from maximum product formation and also the effect of the concentration on enzyme activity when too high, if any. Based on the Enzymatic activity (mol/min) vs Magnesium Chloride concentration (M) graph it can be clearly seen that when the concentration of MgCl2 increases, the enzyme activity increases. It can also be deduced that the optimum concentration of MgCl2 for maximum enzyme activity is higher than 15 mol, which is the highest concentration tested with ( Tube 7 ).

Binding of the substrate to the active site of an enzyme involves interaction with reactive groups provided by the side-chains of amino acids at the binding site. The pH of the incubation medium may affect the ionisation of both the substrate and the amino acid side-chains and will therefore this will affect binding. It may also affect the ionisation of reactive groups that catalyse the reaction, although in the micro-environment of the catalytic site, when it is occupied by the substrate, this is less likely.

Extreme values of pH may also disrupt the tertiary structure of the enzyme, and so distort the active site, or even denature the enzyme protein. As its name indicates, alkaline phosphatase is highly pH-sensitive. In the effective buffering range for Tris-HCl, the initial velocity of the hydrolysis of substrate by alkaline phosphatases hould increases more than 6-fold from pH 7.0 to pH 9.0.

In table 2, it shows that the concentration of Tris-HCl increases with the pH value. However there is drop in concentration value at 0.5min and 1.0 min on pH of 8.5 and at 0.5min on pH of 9. Even the graphs show concentration of Tris-HCl increases linearly except for graph 5 (pH of 8.5) shows inverse linearly proportional graph.The values of the Km and Vmax to be affected by the difference in pH value.

References1. JAKUBOWSKI, (2014).Chapter 7C - Cofactors and Electron Pushing. [online] Employees.csbsju.edu. Available at: http://employees.csbsju.edu/hjakubowski/classes/ch331/catalysis/olelectronpush.htm [Accessed 22 Jun. 2014].2. Brennan, J. (n.d.).How Would the Lack of a Cofactor for an Enzyme Affect the Enzyme's Function? | The Classroom | Synonym. [online] Synonym. Available at: http://classroom.synonym.com/would-lack-cofactor-enzyme-affect-enzymes-function-7502.html [Accessed 22 Jun. 2014]

experiment 3 kinetics

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