SUMMARY

In this experiment, three main objectives were achieved, namely, the quantification of protein, the determination of the protein activity and the determination of the specific binding constant for a protein-receptor pair in two samples (S25 and S60) of unknown protein concentrations. 1) The Fast Protein Liquid Chromatography (FPLC) was used, and print-outs of the concentration of a protein sample were obtained. The protein concentrations were determined by means of a pre-calibrated standard curve. The concentrations of the samples were found to be: Concentration of S25 = 0.144559 mg/ml Concentration of S60 = 0.155822 mg/ml 2) A UV-visible spectrophotometer was used to measure the absorbance and concentration of the reacting mixture at regular intervals of 30 seconds. The protein activity of each sample was determined. One unit of -amylase activity was defined in this experiment to be the amount of enzyme needed to hydrolyze 10 mg of starch per 30 minutes. From the experimental data, the protein activity of -amylase was found to be: Protein activity in S25 = 5.45 units Protein activity in S60 = 4.87 units 3) Using the spectrophotometer, the binding constant of S25 was determined by using constant amounts of S25 to hydrolyze different concentrations of starch. A graph of the reciprocal of rate against the reciprocal of substrate concentration was plotted according to the Lineweaver-Burke equation. The gradient of the straight line obtained was used to determine the binding constant. Specific Binding constant of S25 = 7.76 mg/ml

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Table of Contents
1. INTRODUCTION .................................................................................................................... 1 2. THEORETICAL BACKGROUND ............................................................................................ 3 3. EXPERIMENTAL PROCEDURES......................................................................................... 11 4. RESULTS AND CALCULATIONS ......................................................................................... 14 I. Protein Quantification....................................................................................................... 14 II. Protein Activity ............................................................................................................... 14 III. Specific Binding Constants ............................................................................................ 18 5. DISCUSSIONS ...................................................................................................................... 22 Question 1 ............................................................................................................................ 22 Question 2 ............................................................................................................................ 23 Question 3 ............................................................................................................................ 25 6. ERROR ANALYSIS ............................................................................................................... 27 7. CONCLUSION ...................................................................................................................... 29 8. REFERENCES ...................................................................................................................... 30 9. NOTATIONS ........................................................................................................................ 30 APPENDIX

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1. INTRODUCTION
Starch substances constitute the major part of the human diet for most of the people in the world, as well as many other animals. In order to make use of the carbon and energy stored in starch, the human digestive system, with the help of the enzyme amylases, must first break down the polymer to smaller assimilable sugars, which is eventually converted to the individual basic glucose units. -amylase plays a central role in the complete degradation of starch to metabolisable or fermentable sugars during the germination or malting of cereal grains. Together with starch debranching enzymes, it is also involved in the production of high maltose syrups. -Amylase is usually measured using non-specific reducing sugar assays with starch as substrate. Protein Quantification In this experiment, we are looking into understanding and operating the FPLC system for quantification of proteins. High Performance Liquid Chromatography (FPLC) is a technique widely used for isolating and purifying proteins and peptides. In this experiment, two unknown samples containing -amylase were provided. One sample, S25, was stored at 25C. The other sample, S60, was stored at 60C. Their concentrations were to be determined using Fast Performance Liquid Chromatography (FPLC). Protein Activity Our objective is to determine the activity of -amylase with variation of temperature and protein concentration with the help of a UV spectrometer. Operating the UV spectrometer is necessary for analyzing the relative quantity of starch reacted by the enzyme. Factors such as the speed of a reaction, or how reactive the reactants are, are often areas of interest to chemists. In this experiment, the enzyme -amylase was added to starch and this caused starch to be broken down. Starch concentration was then monitored as time progressed. This allowed us to determine the activity of -amylase. A particular end-point had to be defined for starch hydrolysis by -amylase. In this experiment, it was defined as the time required for the absorbance at 600nm to drop below the value of A600 = 0.4. This value was fixed so that all the concentrations would be based on the concentration when A600 = 0.4, providing a fair basis of comparison. Also, one unit of amylase had been arbitrarily defined in this experiment as the amount of enzyme needed to hydrolyze 10mg of starch per 30 minutes.

Specific Binding Constants An important objective is to optimize the theoretical models such as Lineweaver-Burke plot for determination of the specific binding constant for the protein-receptor pair, which is amylase and starch. In the association reaction between a ligand and a receptor, similar to that of a key and lock function, the binding constant provides a quantitative indication of the affinity of the ligand for the receptor. For this experiment, the value of the binding constant between -amylase and starch was determined. In this experiment, a spectrophotometer was used to determine starch concentrations and by selecting a linearised form of the Michaelis-Menten equation, the binding constant was obtained. The starch was given 8 minutes to be hydrolyzed, and its final concentration was compared with the pre-determined initial concentration. The difference gave the amount of starch that was hydrolyzed in that particular run of the experiment. The slope from the graph of the reciprocal of rate against the reciprocal of substrate concentration gave a value of Km between starch and -amylase.

2. THEORETICAL BACKGROUND
Amylase enzyme Amylase refers to a group of enzymes whose catalytic function is to hydrolyze (breakdown) sugar and starch. It is a digestive enzyme made primarily by the pancreas and salivary glands and can be extracted from soya beans. Amylase digests carbohydrates (polysaccharides) into smaller disaccharide units by catalyzing the hydrolysis of alpha-1,4-glycosidic linkages of polysaccharide to yield monosaccharides such as glucose. Starch iodine complex Starch is made up of amylose and amylopectin. Amylose, the un-branched fraction of native starch, is a polymer of several hundred (1-to-4)-linked glucose units, which in contrast to the linear chain polymer cellulose compound of (1-to-4)-linked glucose adopts a helical conformation. Amylopectin consists of alpha acetal linkage which connects the long chains of glucose units. As a result of the bond angles in the alpha acetal linkage, amylopectin actually forms a spiral much like a coiled spring. A distinct blue color indicates the presence of iodine in solution. Amylopectin in starch is found to be responsible for the formation of the deep blue color in the presence of iodine. The iodine molecule slips inside of the coil present in starch to give the blue-black colour.

Protein Quantification High Performance Liquid Chromatography (FPLC) is a widely used technique to isolate and purify proteins and peptides. In this experiment, a FPLC system, which is similar to that of a FPLC system, is used to determine the protein concentration of two given samples. In liquid chromatography, the protein sample is sent through a chromatographic column. In this column, the sample components will be chased by a mobile phase. Based on the interaction between the components and the column material, there will be different migration rates through the column, for different components. The protein components are separated, eluted, quantified and/or collected depending on the migration rates.

Diagram of FPLC

The main parts of the FPLC are: (a) Pump (b) Injector (c) Column (d) Detector (e) Recording / data processing unit (a) Pump

FPLC pump and solvent reservoir

Beginning with the pump in the FPLC system, it is used to provide pressure so as to force the mobile phase, together with the sample components, through the chromatographic column. 4

When high pressure is applied, the time the mobile phase and sample components spend in the column is decreased. This leads to narrow peaks in the chromatogram, and hence greater sensitivity of the instrument. The mobile phase is forced out from the solvent reservoir using the pump. The pressure gradient provided is around 200 to 400 Pa. (b) Injector It contains a rack for loading of protein samples, which are contained in vials. These protein samples are normally dissolved in the mobile phase first, and injected into the FPLC sample loop via an injection valve. Ideally, the sample should be introduced as a narrow plug into the column so as to reduce broadening of the peak, which increases sensitivity.

Injector unit

(c) Column

Stationary phase

The mobile phase, together with the protein samples will travel into the chromatographic column, containing what is known as the stationary phase. This column is a solid eluting column filled with support through which the mobile phase continuously flows. Ideally, the support should not have any packing irregularities, which might result in channel flows. Channels broaden the peak and also affect sensitivity of the instrument. There will be different migration rates depending on the interactions between the sample components and this stationary phase. (d) Detector The protein sample components finally flow through the stationary phase, pass a detector. The detector consists of a built-in spectrophotometer to measure the absorbance of the eluting compound. The detector is able to measure a response change based on the light of different 5

wavelengths. When the mobile phase flows through it, followed by the mobile phase with protein components, a change is detected and sent to the recording/data processing unit. (e) Recording / data processing unit The mobile phase containing the sample components causes a change in the response of the spectrophotometer, which translates to a peak in the chromatogram (a plot of absorbance versus time). Each compound would have a characteristic peak under certain chromatographic conditions, and the area under the peak is indicative of the concentrations. However, to determine the exact concentration, a calibration curve needs to be created. For this experiment, a standard equilibrium curve of -amylase had been previously prepared with commercial -amylase. The commercial -amylase was then used as a standard to characterize natural-occurring and unstable -amylase. Samples of known concentration of amylase were run through the machine, and graphs consisting of many peaks were obtained. Each of these peak relate to the concentration of the compound added, as seen in Picture 1.

The area under each of these peaks is related to the concentration and can be calculated. Therefore, the linear calibration curve is obtained by plotting the peak area against concentration, as shown in Picture 2.

Samples of unknown concentration (S25 and S60) were injected into the FPLC. The peak area was calculated from the chromatograph and compared with the calibration curve to determine the unknown concentrations. The protein quantity can be calculated from the curves based on Beer-Lamberts law, which states that absorbance of the sample is directly proportional to the concentration of the absorbing sample components.

Protein Activity Different enzymes have different optimum temperatures where they have the maximum activity, as shown in the figure below. At high temperatures, the rate of activity may increase due to additional thermal energy added or thermal denaturation of the enzyme may occur. Thermal denaturation occurs due to the change in the 3-D conformational structure of the enzyme, causing it to lose its catalytic activity.

UV-spectrophotometer The spectrophotometer is used to measure intensity with respect to the wavelength of light. It makes use of UV rays (at 600nm) to determine the absorbance of a sample. A spectrophotometer must be calibrated before it can give concentration readings. This can be carried out by using samples of known concentrations to correlate absorbance and concentration by a linear graph. In this experiment, this was already pre-determined Beer-Lamberts Law In spectrophotometric assays, the concentration of a sample is determined by the amount of light the sample absorbs. Therefore, in utilising spectrophotometry for a quantitative assay, the relationship in the amount of light absorbed and the concentration of the sample must be established. Some of the incident light is absorbed by the solution with concentration c. The incident and transmitted light intensity are then measured and the ratio between them is known as the transmittance (T).

The Beer-Lamberts Law is a combination of the Beers Law and the Lamberts Law. The Beer-Lamberts Law states that the extinction is proportional to the concentration of the absorbing substance (c) and to the thickness of the layer (l) E = cl --- (1) Where E = extinction of the light, expressed as log(1/T) or log( I/I0) (also known as absorption A) = absorptivity or molar extinction coefficient (dm3 mol-1 cm-1) c = molar concentration of the sample (mol dm-3) l = path length of the light (cm)
Absorbing solution concentration, c

Incident light intensity, Io

Transmitted light intensity, I

Light path, l

Specific Binding Constants In general, the enzyme-substrate reaction can be represented by:

where E = Enzyme S = Substrate P = Product concentrations at equilibrium The active site, which is on the enzyme where the substrate binds, comprises only a portion of the overall enzyme structure. The active site is structured in such a way as to form a 3-D 8

structure that is complementary to the shape of the substrate. The substrate will bind itself to the enzyme through hydrogen bonds, salt bridges and van der Waals forces of attraction. Michaelis-Menten Law The kinetics of the reaction is represented by the Michaelis-Menten Law: q = qm[S]/(Km + [S]) where q = reaction rate qm = maximum reaction rate [S] = substrate concentration Km = binding constant The Michaelis-Menten Law could be linearised by some algebraic manipulations to obtain the kinetic parameters qm and Km: (a) Lineweaver-Burke

1 1 Km 1 ( ) q qm qm S
(b) Langmuir
Km S S qm qm q

(c) Eadie-Hofstee
q q = qm Km ( ) S

Data analysis for CII

(1) Significance of the gradient of the concentration vs time graph Gradient =

Concentrat ion Time

The concentration refers to that of unreacted starch (mg/ml) at any point of time. The difference between this concentration and the initial concentration of starch added will give the amount of starch that has reacted. The gradient of the graph will be representative of the rate of starch hydrolysis in this experiment. The trend will also illustrate how the concentration varies with time. The gradient will have units of mg/(ml-min).

(2) Protein activity calculation for S25 and S60 Protein activity in mg/min Protein activity in units = Gradient of concentration-time graph 3mL =A 1 = A = 3A 1 3

Data analysis for CIII (1) Calculation of concentration of starch reacted The concentrations obtained directly from the UV-visible spectrophotometer for each of the initial starch solutions (original and diluted) and after 8 minutes into the progress of the reaction in this part of the experiment were converted to concentration terms in mg/ml. Conc. of starch reacted (mg/ml) Vol. of starch added (ml) Mass of starch reacted (mg) = 10(xi - xf) =3 = 30(xi -xf) where xi, xf: initial, final conc. (%)

(2) Calculation of protein activity in units Rate of reaction, q = = = = Mass of starch reacted / Time of reaction Mass of starch reacted / 8 minutes 30( xi x f ) mg/min 8 90( xi x f ) units 8

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3. EXPERIMENTAL PROCEDURES
This experiment is separated into 3 experiments namely: 1) 2) 3) Protein Quantification Protein Activity Specific Binding Constants

1) Protein Quantification Apparatus FPLC system that has been properly setup and equilibrated 2 samples labeled S25 and S60, which has been stored at 25oC and 60oC respectively Syringe with filter cap and glass container Pipettes

Experimental Set-Up The HPLC system consists of a high pressure pump (mostly two, a supply of mobile phases(s), a column containing the stationary phase, an injection unit for introducing the samples onto the column, an in-line detector and some method of displaying the detector signal. The choice of the stationary and the mobile phases depends on the proteins in the mixture, and the sequence of the chromatographic technique in the downstream processing train.

Block Diagram of an HPLC System

11

Procedures 1) The two unknown samples (each containing a different concentration of -amylase) are labeled S25 and S60. Each was stored at 25C and 60C respectively for two hours before the commencement of the experiment. Using a pipette, extract 20l of S25 and place it into a syringe. Attach the filter onto the nozzle of the syringe. Depress the plunger of the syringe to inject the S25 sample into the glass bottle. Place the glass bottle into the well of the injection unit of the FPLC system. Start the system and wait for the analysis to be completed. Print the data and the graphs. Concentration of the sample was calculated by the software and is represented by the area under the curve. Repeat steps 2-7 using sample S60.

2) 3) 4) 5) 6) 7) 8)

2) Protein Activity Apparatus UV-visible spectrophotometer with wavelength set at 600nm Test tubes 4 Curettes Micro-pipettes Distilled water 250ml of 4mg/ml starch solution 200ml of 0.5mM iodine solution 2 unknown samples labeled S25 and S50, which had been stored at room temperature and at 60 C for 2 hours respectively

Procedures 1) 2) 3) 5 ml of iodine solution was pipetted into each of the 10 test tubes. 3 ml of the starch solution(0.4% starch) was pipetted into a test tube. Prepare a control to be used as blank for the absorbance measurements by pipetting 1ml of the S25 solution into 3mL distilled water. Ass 0.2mL of this to a test tube containing iodine. At t = 0 s, 1mL of S25 was added to the starch solution. At 30 seconds intervals, 0.2 ml of this reaction mixture was added into the test tubes containing iodine. The solution in the test tubes containing the reaction mixture and the iodine is then extracted and placed into the curettes to be tested using the UV-visible spectrophotometer. The absorbance and concentration was then measured against the control prepared in step 3. The end-point of starch hydrolysis was defined as the time to reach A600 = 0.4. This time had to be in the interval between 3 and 10 mins. Repeat steps 1-7 for S60. 12

4) 5) 6)

7)

3) Specific Binding Constants Apparatus UV-visible spectrophotometer with wavelength set at 600nm. Micro-pipettes 4 Curettes Test tubes Starch solutions of 5 pre-determined concentrations (3%, 2%, 1.5%, 1.0%, 0.75%). 0.5 mM Iodine solution. A solution of S25

Experimental Procedure 1) 2) 3) 4) 5) 6) 7) 8) 9) 10) 5 mL of iodine solution was pipetted into each of the 5 test tubes. Prepare the starch solutions of concentrations 3%, 2%, 1.5%, 1.0% and 0.75% from the stock solution of 3% starch. To prepare a 2% starch solution, extract 2mL of the 3% starch solution into a test tube and to it, add 1mL of distilled water. To prepare a 1.5% starch solution, extract 1.5mL of the 3% starch solution into a test tube and to it, add 1.5mL of distilled water. To prepare a 1% starch solution, extract 1mL of the 3% starch solution into a test tube and to it, add 2mL of distilled water. To prepare a 0.75% starch solution, extract 0.75mL of the 3% starch solution into a test tube and to it, add 2.25mL of distilled water. Place 3mL of the starch solution prepared from steps 2-6 into each of the 5 test tubes. At t = 0 s, 1 mL sample of S25 was added to each of the 5 test tubes containing starch. After 8 minutes, 0.2 mL of the reaction mixture was pipetted into each of the 5 test tubes containing iodine solution. The absorbance at 600nm was measured using the spectrophotometer. The solution in the test tubes containing the reaction mixture and the iodine is then extracted and placed into the curettes to be tested using the UV-visible spectrophotometer. A control need not be prepared since the spectrophotometer has already been calibrated from the previous steps of the experiment.

11)

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4. RESULTS AND CALCULATIONS

I. Protein Quantification
To determine the concentration of the unknown samples S25 and S60, each of them was fed into the FPLC column. The peak area under the curve obtained for each sample was computed and the unknown concentration for each sample was obtained using the calibration curve. The chromatogram curves for S25 and S60 are attached in the Appendix. Concentration of S25 = 144.559 ppm = 0.144559 mg/ml Concentration of S60 = 155.822 ppm = 0.155822 mg/ml

II. Protein Activity

The reaction mixture was quenched and added into the 5 ml iodine solutions in the 20 test tubes at 0.5 min intervals. This was done to stop the protein activity so as to determine the absorbance of light in the UV-visible spectrophotometer with wavelength set at 600 nm. The proportion of starch remaining in the reaction mixture corresponds to the absorbance of light measured, which was relative to the blank sample prepared containing -amylase solution and distilled water. The end point of starch hydrolysis (tabs=0.4) is defined as the time to reach A600 = 0.4. Given values: Conc. of starch 1 unit of -amylase activity = 0.4 % = 4 mg/ml = 10 mg of starch / 30 min = mg of starch/min Vol. of starch solution (0.4 % starch) = 3 ml Sample S25 Time (min) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 Absorbance 0.662 0.558 0.523 0.449 0.435 0.423 0.349 0.336 0.318 0.306 Concentration (%) 0.3533 0.2975 0.2792 0.2395 0.2321 0.2256 0.1864 0.1794 0.1699 0.1634

Table 1 : Absorbance and Concentration for S25 at 0.5 min intervals

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Graph of Absorbance vs Time (min) [S25]

0.75 0.65 Absorbance 0.55 0.45 0.35 0.25 0 1 2 Time (min) Figure 1 : Graph of Absorbance vs Time for S25 3 4 5

Graph of Absorbance vs Concentration (%) [S25]

0.7 0.65 0.6 0.55 0.5 0.45 0.4 0.35 0.3 0.15 0.2 y = 1.8762x - 0.0005 R = 1

Absorbance

0.25

0.3

0.35

0.4

Concentration (%) Figure 2 : Graph of Absorbance vs Concentration for S25

From Fig. 1, tabs=0.4 = 3.0819 min From Fig. 2, Conc.abs=0.4 = 0.21346 % Amount of starch hydrolysed Reaction rate of starch Protein activity of S25 = (4 2.1346) X 3 = 5.5961 mg = 5.5961 / 3.0819 = 1.8158 mg/min = 1.8158 / ( ) = 5.4474 5.45 units 15

Sample S60

Time (min) 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0

Absorbance 0.688 0.635 0.584 0.504 0.468 0.426 0.398 0.349 0.364 0.343 0.382 0.313 0.346 0.337

Concentration (%) 0.3673 0.3385 0.3116 0.2688 0.2496 0.2275 0.2121 0.1861 0.1941 0.1827 0.2037 0.1670 0.1844 0.1797

Table 2 : Absorbance and Concentration of S60 at 0.5 min intervals

Graph of Absorbance vs Time (min) [S60]

1 0.8 Absorbance 0.6 0.4 0.2 0 0 1 2 3 4 Time (min) Figure 3 : Graph of Absorbance vs Time for S60 5 6 7 8

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Graph of Absorbance vs Concentration (%) [S60]

0.8 0.7 Absorbance 0.6 0.5 0.4 0.3 0.15 0.2 0.25 0.3 0.35 0.4 Concentration (%) Figure 4 : Graph of Absorbance vs Concentration for S60 y = 1.8728x + 0.0005 R = 1

From Fig. 3, tabs=0.4 = 3.4490 min From Fig. 4, Conc.abs=0.4 = 0.21332 % Amount of starch hydrolysed Reaction rate of starch Protein activity of S60 = (4 2.1332) X 3 = 5.6004 mg = 5.6004 / 3.4490 = 1.6238 mg/min = 1.6238 / ( ) = 4.8713 4.87 units

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III. Specific Binding Constants

The Michaelis-Menten equation can be linearised into the following kinetic parameters: (a) Lineweaver - Burke (b) Langmuir (c) Eadie Hofstee

(a) Lineweaver-Burke equation

1 1 Km 1 q qm qm S
q=-( where [S]f : substrate final concentration [S]i : substrate initial concentration S : substrate concentration (% or mg/ml) q : reaction rate (mg/ml-min) qm : maximum reaction rate (mg/ml-min) Km : binding constant (% or mg/ml) Plot of 1/q against 1/S will yield a straight line with gradient = Km/qm and Y-intercept = 1/qm. )

[S]i (%)

Absorbance

[S]f (%)

1/[S]i

q (mg/ml-min)

1/q (ml-min/mg)

3 2 1.5 1 0.75

2.232 1.237 0.885 0.491 0.337

1.1907 0.6597 0.4722 0.2618 0.1800

0.333 0.500 0.667 1.000 1.333

0.226163 0.167538 0.128475 0.092275 0.07125

4.4216 5.968813 7.783615 10.83717 14.03509

Table 3 : Calculated values for Lineweaver-Burke equation

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Graph of 1/q vs 1/[S]i

16 14 1/q (ml-min/mg) 12 10 8 6 4 2 0 0.2 0.4 0.6 0.8 1/[S]i Figure 5 : Graph of 1/q vs 1/[S]i for Lineweaver-Burke equation 1 1.2 1.4 y = 9.6142x + 1.2384 R = 0.9996

From Fig. 5, Y-intercept = 1.2384 = 1/qm => qm = 0.80749 0.807 mg/ml-min Gradient = 9.6142 = Km / qm => Km = specific binding constant = 7.7634 7.76 mg/ml (b) Langmuir equation

S Km 1 S q qm qm
Plot of S/q against S will yield a straight line with gradient = 1 / qm and Y-intercept = Km / qm.

[S]i (%)

Absorbance

[S]f (%)

q (mg/ml-min)

[S]i / q (min)

3 2 1.5 1 0.75

2.232 1.237 0.885 0.491 0.337

1.1907 0.6597 0.4722 0.2618 0.1800

0.226163 0.167538 0.128475 0.092275 0.07125

13.26477 11.93759 11.67542 10.83717 10.52632

Table 4 : Calculated values for Langmuir equation

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Graph of [S]i/q vs [S]i

13.5 13 [S]i / q (min) 12.5 12 11.5 11 10.5 10 0.5 1 1.5 2 [S]i Figure 6 : Graph of [S]i/q vs [S]i for Langmuir equation 2.5 3 3.5 y = 1.1926x + 9.6804 R = 0.9864

From Fig. 6, Gradient = 1.1926 = 1 / qm Y-intercept = 9.6804 = Km / qm

=> qm = 0.83850 0.839 mg/ml-min => Km = specific binding constant = 8.1171 8.12 mg/ml

(c) Eadie Hofstee equation

q qm K m

q S

Plot of q vs q/S will yield a straight line with gradient = -Km and Y-intercept = qm.

[S]i (%)

Absorbance

[S]f (%)

q (mg/ml-min)

q/[S]i (1/min)

3 2 1.5 1 0.75

2.232 1.237 0.885 0.491 0.337

1.1907 0.6597 0.4722 0.2618 0.1800

0.226163 0.167538 0.128475 0.092275 0.07125

0.075388 0.083769 0.08565 0.092275 0.095

Table 5 : Calculated values for Eadie-Hofstee equation

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Graph of q vs q/[S]i
0.25 0.23 0.21 0.19 0.17 0.15 0.13 0.11 0.09 0.07 0.05 0.07 0.075 0.08 0.085 q/[S]i (1/min) Figure 7 : Graph of q vs q/[S]i for Eadie-Hofstee equation q (mg/ml-min)

y = -7.9419x + 0.8235 R = 0.9794

0.09

0.095

0.1

From Fig. 7, Y-intercept = qm = 0.8235 mg/ml-min Gradient = -7.9419 = -Km => Km = 7.9419 mg/ml From Figs. 5-7, which represent the linear plots of the Lineweaver-Burke equation, Langmuir equation and the Eadie-Hofstee equation respectively, it can be seen that the LineweaverBurke plot in Fig. 5 has the R2 value closest to 1, which implies that it is the most accurate. Therefore, the Lineweaver-Burke equation is selected as the linearised form of MichaelisMenten relation.

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5. DISCUSSIONS
Question 1
Briefly describe the experiment that you designed in CI. What are the protein concentrations in S25 and S60 in mg/mL? In this experiment, the Fast Performance Liquid Chromatography (FPLC) is used. Before the experiment, the FPLC is well calibrated using a standard curve. The standard curve is obtained using the chromatogram graph for a series of known concentrations of the standard solution -amylase. -amylase is used as the standard to characterize natural-occurring and unstable -amylase used in this experiment. After the proteins are eluted, the area under each peak of the chromatogram graph are calculated and tabulated and these are used to form the points for the calibration curve. For this experiment, a standard calibration curve of -amylase had been prepared and the information was readily available in the FPLC. In the experiment, 1ml of sample S25 / S60, of which concentrations were unknown, was pipetted into a syringe and inject into a vial through filter paper. The filter paper helped to minimize contamination and the process was carried out carefully to prevent any air bubbles from collecting in the vial. The vial was placed in the FPLC and the samples concentration was analyzed through the computer. According to the Beer-Lamberts Law: E = cl Where E c l = = = = extinction of the light / absorption A absorptivity or molar extinction coefficient (dm3/mol-cm) molar concentration of the sample (mol/dm3) path length of the light (cm)

we can deduce that there is a linear relationship between E and . When S25 or S60, with unknown concentrations, were tested in the FPLC, the areas under the peak of their chromatograms were compared with the standard calibrated curve to determine their concentrations. Eventually, the concentrations of S25 and S60 are 0.144559 mg/ml and 0.155822 mg/ml respectively. The concentration readings should have been the same since they were identical samples. However, the slight deviation could be due to experimental errors, which will be further elaborated under Error Analysis.

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Question 2
Based on the end-points of starch hydrolysis in CII, determine the protein activity present in S25 and S60. Account for any differences, and discuss the significance of protein activity vis-a-vis protein concentration (answers in Q1) with regards to pharmaceutical drug assay. Sample Concentration (mg/ml) Protein activity (units) S25 2.1346 5.45 S60 2.1332 4.87

Table 6 : Concentration and activity of S25 / S60

From Table 6, it can be seen that the protein activity of S25 is higher than that of S60. This difference in activity could be due to the difference in temperature in which the two samples were stored at. As most enzymes have an optimum temperature as seen in Fig. 8, at which the activity is the maximum, high temperatures would reduce the enzyme reaction rate because the bonds which maintain the enzymes specific structure are broken and this denatures the enzyme.

Figure 8 : Rate of reaction for enzymes

Therefore, the enzymes in the S60 sample, which was stored at 600C for 2 hours, should have been denatured under the high temperatures and affect its catalytic ability. This explains why S60 has a lower activity as compared to that of S25. Moreover, from Fig.5, there is a positive relation between the enzyme concentration and rate of reaction. This is because as the enzyme concentration increases, the number of available substrate sites increases proportionally. Therefore, the reaction rate, which is also reflected by the protein activity, will increase correspondingly. From the discussion above, we can deduce that both temperature and concentration are possible variables which affect the enzymes activity. To have a more accurate method of comparing the change in activity of the sample with respect to concentration, specific activity, which is the activity divided by the concentration, can be used. The specific activity of S25 is higher than that of S60. Specific activity of S25 = 5.4474 units / (2.1346 mg/ml) = 2.55 units/mg Specific activity of S60 = 4.8713 units / (2.1332 mg/ml) = 2.28 units/mg 23

Some other factors which may affect protein activity are: (1) Ph value of the solution (2) Presence of co-factor or inhibitors (3) Buffer ions A drug assay is carried out to determine the concentration of a component in a mixture. Drugs inhibit enzymes by the binding of ligands to a target protein. Therefore, knowing the exact structure of the target protein of a particular disease will allow researchers to create drugs that are designed to fit the proteins structure to prevent the disease from displaying its harmful characteristics. From the experiment, S25 has a higher activity than S60. This shows that activity is affected by temperature and is thus essential to find the optimal temperature for the drugs to be the most efficient. Protein concentration is defined as the amount of protein per unit volume of solution and its activity is the amount of substrate converted per unit time. With regards to pharmaceutical drug assay, protein concentration refers to the amount of protein of the disease per unit volume and its activity refers to the amount of ligand in the drugs converted by the protein of the disease per unit time.

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Question 3
Explain your choice of the linearised form of the Michaelis-Menten relation in CIII. Briefly explain the experiment that you designed in reference to this choice. Determine the binding constant between starch and -amylase in units of A600. Choice of linearised form of the Michaelis-Menten relation As seen from CIII in the Results and Calculations section, the Michaelis-Menten equation can be linearised into 3 different equation, mainly the Lineweaver-Burke equation, Langmuir equation and Eadie-Hofstee equation. Below are the advantages and disadvantages of the 3 different linear equations. (a) Lineweaver-Burke equation

1 1 Km 1 q qm qm S
A double-reciprocal plot of 1/q vs 1/S will allow more accurate determination of qm and Km because it increases the significance of very low substrate concentrations, which may have an overall effect on the values of qm and Km. The x-axis (1/S) is independent of the y-axis (1/q). This means that errors in either axis would not affect the other axis. Most common line equation used by biochemist in analyzing the behaviour of enzyme under influence of inhibitor or accelerator. Allows easy identification of important terms in enzyme kinetics, such as Km and qm.

(b) Langmuir equation

S Km 1 S q qm qm
Inter-dependence of the x-axis (S) and the y-axis (S/q) will lead to a larger increase in percentage error of the calculated qm and Km. Mostly used for solution-solid adsorption data and estimation of the adsorption equilibrium constants. Useful for moderate high concentrations. However, for this experiment, concentrations are relatively low.

(c) Eadie-Hofstee equation

q qm K m

q S

Inter-dependence of the x-axis (q/S) and the y-axis (q) will lead to a larger increase in percentage error of the calculated qm and Km.

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Moreover, comparing the R2 values:

Linear equations Lineweaver-Burke (Fig. 5) Langmuir (Fig. 6) Eadie-Hofstee (Fig. 7) R2 0.9996 0.9864 0.9794

Table 7 : R2 values for the different linearised forms of the Michaelis-Menten relation

It can be seen that the R2 value is the closest to 1 for Lineweaver-Burke equation. Therefore, together with the reasons mentioned above, we have selected the Lineweaver-Burke equation as the linearised form of the Michaelis-Menten relation. Km (Lineweaver-Burke) = 7.76 mg/ml Determine Km in units of A600 From Fig. 2, Absorbance (A600) = substrate concentration (%) X 1.8762 0.0005 Km (in units of A600) = 0.77634 X 1.8762 0.0005 = 1.4561 1.46 Brief explanation of experimental design in reference to the Lineweaver-Burke equation The experimental procedures have been mentioned earlier in the report under the Experimental Procedure section. As the Lineweaver-Burke equation is a linear relation between 1/q and 1/S, a minimum of 5 points are required to plot the graph. Therefore, we are required to prepare 5 pre-determined starch concentrations from the 3% starch provided. Table 7 shows the dilution amount that our group has decided on to come up with the 5 different pre-determined starch concentrations.
Starch (%) 3 2 1.5 1 0.75 Amount of Starch (ml) 3 2 1.5 1 0.75 Amount of De-ionised water (ml) 0 1 1.5 2 2.25

Table 8 : Preparation of 5 pre-determined starch concentrations

After preparing these 5 different starch concentrations, proceeding along the experiment will yield data as shown in Table 3. Using the data from Table 3, we are able to plot the Lineweaver-Burke plot to determine the values of Km and qm.

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6. ERROR ANALYSIS
1) During the testing for protein activity, when 0.2ml of the reaction mixture, consisting of the 0.4% starch and the sample -amylase, was extracted and added to the iodine solution at 30 seconds interval, the pipetting tip must be changed before extraction at every time interval. This is to prevent contamination of the samples because as time progresses, the starch will be broken down and therefore the starch concentration will differ. 2) When pipetting, one must make sure that there are no bubbles present in the pipetting tip. This is because air bubbles in the tip will affect the accuracy of the pipette. Therefore, in the event of an air bubble trapped in the tip, the tip must be changed. 3) While using the pipette, one must be careful of the extent of depressing the plunger of the pipette. This is because, if the plunger is pressed beyond the first point of resistance, more than stated amount of solution will be extracted by the pipette. Also, to prevent bubbles from forming when extracting the solution, the process of depressing and releasing the plunger must be done slowly. 4) The tips used for one of the pipette was not a single-use disposable tip; Rather, one tip was used to extract one particular solution each, throughout the entire experiment. However, while not in use, the other tips were all placed together in a beaker. As a result, the apex of the pipette tips were all in contact with each other and this may lead to contamination of the tip while it is in use. Therefore, to prevent contamination, the tips should be stored separately. 5) Before extracting any solution, the solution should be shaken to ensure that the solution is homogenous. For instance, for the process of protein quantification, our experimental data deferred vastly from one another (concentration of S25 was 144.559ppm, while the concentration of S60 was 155.822ppm) when it should not have any differences. This error in the result may have been because prior to extracting the samples, the amylase solution was not well mixed enough to ensure homogeneity within the solution. Therefore, in order to reduce such errors, all solution must be mixed well to ensure homogeneity. 6) In order to reduce human error, one person should be in charge of doing the pipetting so that the usage of the pipette is standardized throughout the experiment. 7) During the process of extracting and adding the reaction mixture to the iodine at different time intervals, human reaction time would have had contributed to experimental errors. 8) During the preparation of the samples for the use of the HPLC system, 1ml of the sample was injected into the bottle to be placed within the HPLC unit, via a syringe; And attached to the syringe was a filter which was used to remove any tiny particles that could cause the HPLC system to clog. When injecting the sample solution into the bottle, one must 27

do it slowly and carefully so as to prevent the formation of bubbles in the bottle. 9) For the S60 samples, it was heated to 60 oC for 2 hours before usage. However, through the experiment, the sample was not at 60 oC, rather, it was at room temperature. As a result, the protein activity obtained for the S60 sample would not have been accurate. 10) During the usage of the UV-visible spectrophotometer, the side of the cuvette exposed to the UV light may not have been completely cleaned or free of scratches. As a result, the full intensity of the UV may not have completely passed through the solution. Therefore contributing to errors in the experimental data. 11) Only 4 curettes were available for use, as such, they had to be repeatedly washed and dried. In the event where the curette is not dried properly, it would lead to inaccuracies in the experimental data. 12) The plotting of the graph to obtain the specific binding constant used only 5 data points to plot a best fit line. As a result, the Km value obtained might be inaccurate. Therefore, more data points should be obtained for a more accurate Km value. Precautions 1) Ensure that the cuvettes used are thoroughly washed and dried. Also, ensure that the surfaces of the cuvettes are free of fingerprints, scratches or marks. 2) Shake the solutions to ensure homogeneity before extraction.

3) After starch has been added to the test tubes, ensure that the test tubes are shaken thoroughly. This is to ensure that the reaction has taken place thoroughly.

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7. CONCLUSION
Through the first part of the experiment, protein quantification, the objectives of identifying and using the HPLC system was met and the concentrations of the S25 and S60 samples were found to be 144.559 ppm and 155.822 ppm respectively. While the concentrations of both samples were expected to be the same, our experimental results differed from the expected results due to possible experimental errors. While for the second section of the experiment, protein activity was determined using the UV-visible spectrophotometer and found to be 5.45 units for the S25 sample and 4.87 units for the S60 sample. In general, it can be concluded from the experimental data that the sample subjected to a higher temperature (S60) showed a lower activity. This may be due to possible denaturation occurring during the heating of the sample. The third section of the experiment required us to experimentally determine the specific binding constants with the use of the linearised forms of the Michaelis-Menten relation, namely the Lineweaver-Burke equation. The plot of the Lineweaver-Burke equation found the value of the specific binding constant, Km to be 7.76 mg/ml.

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8. REFERENCES
1. Bailey, J. E. and Ollis. D. F. Biochemical Engineering Fundamentals, 2nd Ed., Chapter 3, McGraw-Hill, 1986. 2. Chromatography- Theory, FPLC and Beyond. Retrieved on 18, March, 2010, from: http://www.mnstate.edu/biotech/chrom_fplc.pdf 3. Enzyme Kinetics. (2003). Retrieved February 29, 2008, http://users.rcn.com/jkimball.ma.ultranet/BiologyPages/E/EnzymeKinetics.html from:

9. NOTATIONS
A A600 c Km l q qm S S25 S60 -

Absorbance Absorbance with wavelength set at 600nm Concentrations of the absorbing species in the material Binding constant Distance that the light travels through the material or the path length Reaction rate Maximum reaction rate Substrate concentration Sample containing -amylase at 25C Sample containing -amylase at 60C

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APPENDIX

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