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Assignments Due

• Begin a Materials and Methods section that includes the procedures through Day 5; include appropriate References (cited as in Protein Expression & Purification). Bring a TYPED draft to Day 6 lab for evaluation.
  See Research Paper .
• Results (McMillan: pp. 30-50, 66-71, 81-83, 126-160 (3rd ed.); 45-67, 83-89, 108-109, 167-205 (4th ed.))
  See Research Paper .
• Review enzyme kinetics. Consult any general biochemistry textbook. Understand Km, Vm, types of inhibition and methods of graphing the results.
  
• Review Using figures (graphs), Examples of graphs (from Experimental Biosciences Resources)

Preparation

• Writing the Results
• Spectrophotometry
• Kinetics review

Overview of Experiment

Kinetic constants for the purified adenosine deaminase will be determined for two substrates and an inhibitor. Each team will use their purified enzyme preparations for these studies. Km data should be compared within a section to determine if the native and recombinant enzymes have the same kinetic properties.

We will be using a wavelength of 235 nm for the kinetic assays.

NOTE re raw data: 1/2 of each team will work with the recombinant sample, and the other 1/2 will work with the native; you must record ALL of the raw data for BOTH sources in your lab notebook during this lab session.

Background

Adenosine deaminase, ADA (Adenosine aminohydrolase, EC 3.5.4.4), reacts with adenosine or several adenine nucleoside analogs catalyzing the irreversible reaction:

adenosine + H2O NH4 + inosine.

The reaction scheme for this enzyme reduces to that of the most simple enzyme catalyzed reaction:

(Scheme 1).

E represents the enzyme, S is the substrate (adenosine), ES is the enzyme substrate complex, and P is the product. Because the reaction is performed in an aqueous solution, water concentration is saturating and does not contribute to the rate equation. This scheme is also valid for multisubstrate reactions when all the other substrates are held at saturating concentrations.

Measurements of the velocity of the reaction in the absence of products allow determination of kinetic parameters. These parameters aid in comparison of the properties of the enzyme from different species and sources. The kinetic constants are also useful for analyzing alternative substrates. The most universal parameters are known as the Michaelis-Menten constants.

maximum velocity: Vm = k3[Etotal]

"substrate affinity" constant: Km = (k2+k3) / k1

For multisubstrate and more complex binding schemes the Km is not as simple. In any case, the Km is defined experimentally as the substrate concentration required to give half maximal activity.

For Scheme 1 the rate equation is:

(2)

or inverted:

(3)

where
v= measured initial reaction velocity
Vm= maximum reaction rate for that enzyme concentration
[S] = substrate concentration
Km= Michaelis constant defined above

Because Vm is directly proportional to the enzyme concentration, a level of enzyme must be found that will produce a reaction rate that is conveniently measured. Careful choice of enzyme levels is also important to ensure that the level of substrate in the assay solution remains constant over the course of the measurement. It is wise to calculate the nmoles of substrate in the assay solution and determine that this amount does not vary by more than 10% during the course of the measured reaction. The first step of this experiment will determine a suitable level of adenosine deaminase to be used in the kinetic studies.

The assay for this enzyme is derived by looking at the UV absorption spectra of inosine and adenosine and determining the wavelength at which the difference in their absorbance is the greatest or most suitable (see "Absorbance Spectra" below). For our experiment, the wavelength of 235nm is chosen so that saturating levels of adenosine can be used. At this wavelength, an increasein absorbance is seen as adenosine is converted to inosine. The slope is opposite the 265nm assays that you used previously. The assays are completed using similar spectrophotometer instructions as for the previous assays.

In addition to adenosine, the deaminase can catalyze reactions with 2'-deoxyadenosine, 2'-fluoroadenosine, 2',6'-diaminopurine riboside, and 3'-deoxyadenosine and will dehalogenate several N6-haloadenosine compounds. Each substrate binds with different affinity and is converted to product at different rates resulting in different Km's and/or Vm's. In Part 2 of this experiment, the kinetic parameters of adenosine and 2'-deoxyadenosine will be compared as a measure of the influence of the sugar group of the substrate on the binding affinity and reactivity of the substrate.

An inhibitor of the adenosine deaminase reaction will be evaluated in Part 3 of this experiment. The type of inhibition, competitive, noncompetitive, or uncompetitive, will be determined and a Ki will be calculated.

To demonstrate how a wavelength for a particular reaction is chosen and how an extinction coefficient is calculated, ABSORBANCE SCANS of substrate (adenosine) and product (inosine) are presented. Overlays of consecutive scans of both compounds are shown. Compare the absorbance spectra of adenosine and inosine at 265 nm (where we've performed previous ADA assays) versus 235 nm (where we'll perform ADA kinetic assays). As a class, we are going to calculate the EXTINCTION COEFFICIENT at 235 nm; you will use this value in your kinetic assays to convert A/min to nmol/min.

Consider the following:

1. Why is 265nm used in published assays of this enzyme?
2. What is an advantage to using 235 nm for your assays today?
3. What was compromised by changing the wavelength?

****SUPPLEMENTAL PROCEDURE INFORMATION: Determining the Molar Extinction Coefficient for the Reaction at 235 nm from the Absorbance Spectra****

Place cuvettes containing phosphate buffer in the rear and front compartments of the Cary 118 spectrophotometer.
a) Record a baseline scan from 350nm to 220nm at a rate of 1nm/sec and chart speed of 20 sec/inch.
b) Reverse the scan direction and bring the pen back to the starting point. Do not adjust any settings on the instrument at this point.
c) Replace the phosphate buffer in the cuvette nearer to the front of the instrument with a nucleoside solution and repeat the scan using a different color ink pen in the recorder.
Repeat steps b) and c) with the other nucleoside.

Procedures

Purpose: To familiarize the student with the concepts and principles of simple kinetic determinations. Specifically you will: 1) determine appropriate spectrophotometric assay parameters, 2) determine a suitable concentration of enzyme for kinetic studies, 3) determine Km's and Vm's from saturation curves , and 4) evaluate inhibition patterns and calculate Ki's from double reciprocal plots.

EACH TEAM MUST REPORT THEIR Km VALUES FOR BOTH SUBSTRATES FOR BOTH NATIVE and RECOMBINANT ADA to the Instructor before proceeding with Part 3.

NOTE: The Factor is a number you enter for the instrument to use in converting ΔA/min to the desired units (e.g., nmol/min); the derivation of the factor must appear in your notebook.

1) Genesys 5 spectrophotometer

• Turn on the instrument.
• After the instrument has completed its initialization and calibrating routine, select 6. simple kinetics.
• Press TEST TYPES and choose the program "ADA_KIN."
• Display the settings used in this program to record in your notebook by pressing the "SET UP TEST" key. The settings continue on the next page which is viewed by pressing the soft key located below the next pagedisplay at the bottom of the LCD screen.
• Enter the number for the Factor and SAVE the program to retain the number that was just entered.
  DO NOT CHANGE ANY OF THE OTHER SETTINGS.
• EXIT to the simple kinetics window (graph appears). At this point you can place your sample in position (or cell) 2 and begin recording rates. By default, the instrument will zero against "air" in the first cuvette position at the beginning of each measurement. When measuring the change in absorbance for these rate determinations, the absolute zero is not as critical as when doing quantitation by single readings such as A280's.
• Perform the BLANK RUN and ADA assay procedures using the volumes of buffer and adenosine specified in Part 1 (NOTE: you do NOT need to use a CONTROL enzyme).

2) Biowave S2100 UV/Vis Diode Array spectrophotometer

• Turn on the instrument.
• After the spectrophotometer has performed the self-diagnostics press the "Cont." key to proceed to the first menu screen.
• Press the "Other" key followed by the "Other methods" key to access the pre-programmed routines.
• Select "ADA KINETICS" and then click on "Accept."
• Record the program settings in your notebook.
• Press "Program".
• Use the arrow to select "Factor".
• Press "change" and enter the NUMBER for the Factor.
• Press "accept" and then "all ok".
• Press "Run"; the instrument is now ready to obtain measurements at the settings defined.
• Perform the REF and TEST (BLANK RUN and ADA assays) procedures using the volumes of buffer and adenosine specified in Part 1 (NOTE: you do NOT need to use a CONTROL enzyme).

3) Libra S22 spectrophotometer

• Turn on the instrument.
  
• After the spectrophotometer has performed the self-diagnostics, press "2" on keypad to enter Applications.
• Press "3" to select Reaction Rate.
• Set wavelength to 235 nm and press F3 ("OK").
• Press "1" to select seconds and press F3 ("OK").
• Enter 10 for Delay Time and press F3 ("OK").
• Enter 60 for Duration and press F3 ("OK").
• Enter Factor that you calculated and press F3 ("OK").
• Record the program settings in your notebook.
• Press F3 to select Run; the instrument is now ready to obtain measurements at the settings defined.
• Perform the REF and RUN (BLANK RUN and ADA assays) procedures using the volumes of buffer and adenosine specified in Part 1 (NOTE: you do NOT need to use a CONTROL enzyme).

Reagents and Supplies

0.05 M potassium phosphate, pH 7.4
adenosine deaminase (your most pure sample)
3 mM adenosine (in KH2PO4 buffer)  NOTE: please take only 10-15 ml so there's enough for the entire class
3 mM 2'-deoxyadenosine (in KH2PO4 buffer)
3 mM N6-methyladenosine (in KH2PO4 buffer)

NOTE: if you want to save time do NOT print each graph; you cannot do anything else with the specs while printing.
I recommend either not printing at all OR printing just a few "sample" graphs that are representative of your study.

Part 1: Level of Adenosine Deaminase to Use in the Kinetic Assays

Note: Kinetic studies are difficult to perform successfully. It is imperative that measuring and pipetting be done with care and precision to accomplish the accuracy required for these studies.

Find the amount of enzyme to use in the kinetic assays by completing the following assays. Prepare 3 ml of assay solutions according to the following table and record the absorbance changes at 235nm. (The volumes of ADA given are typical but you may need to expand the range if your activity is dilute OR dilute your sample in ADA assay buffer if it's highly active. Use the assay results from Day 4 to get a rough estimate for an appropriate range for your sample and adjust the buffer volume if necessary.)

It will be necessary to find a suitable volume of your stock enzyme solution that produces a ΔA₂₃₅/min of 0.02-0.03 under conditions of saturating levels of substrate as found in the above solutions. Construct a graph in your notebook of ΔA/min versus amount of enzyme (µl) used and determine the volume of enzyme to use for the remaining kinetic measurements. You may round the calculated volumes to a convenient volume (e.g., 10, 15, 20, etc. µl).
This graph should demonstrate that the reaction rate is linearly dependent on the level of enzyme at saturating levels of substrate. If your plot is not linear, see the instructor for further instructions.

Part 2: Km's of Adenosine and 2'-Deoxyadenosine

To determine Km and Vm for a particular substrate, the rate (or velocity) of the reaction is measured at various concentrations of the substrate.
[The amount of enzyme and the total volume of the assay solution must be held constant ±2% of the total volume. Note that in Part I the total volume of the assay solution varied by 35 µl. This change represents less than 2% of the total solution and normal pipetting errors are expected to be about 3% due to multiple additions. Therefore, these small volume changes are within the experimental error and will not make a significant contribution to the total error.] One method to achieve varied concentration is to add different volumes of a stock solution of the substrate and adjust the volume of buffer to maintain the constant volume of the reaction mixture. We will use this method for kinetic determinations using adenosine and 2'-deoxyadenosine.

Construct a chart in your notebook of the volumes of each component added to the reaction mixture so that the total volume is 3 ml. Measure the reaction velocities with 0.005, 0.010, 0.020, 0.040, 0.080, 0.10, 0.30 ml of adenosine solution. Compare the reaction rates of the 0.1 ml and the 0.3 ml adenosine assays. If these rates are not within 10% of each other, try 0.35 ml of adenosine. A graph of velocity (nmol/min) versus [adenosine] (µM) should show that an asymptote is being approached. Repeat the assays using 2'-deoxyadenosine and plot the data on the SAME graph.

From the plot of velocity versus substrate concentration (µM) estimate the Km for adenosine to use in Part 3. (See Results examples at the end of this section.) BEFORE beginning Part 3, confirm with the Instructor or TA that your number is reasonable based on literature values for the enzyme.

Part 3: Inhibition of Adenosine Deaminase Activity

NOTE: Timely completion of this work reflects sufficient preparation, good team work, and efficient time management. For practical purposes, if you are NOT actually RUNNING assays by 3:00 p.m., you will not be able to complete the lab session and will have to get the inhibition data from someone else.

You have several hours to complete Parts 1 & 2 and prepare the dilutions for Part 3; if you are still working on Parts 1 & 2 at 3 p.m., that means you have had major experimental difficulties--at this point, it's better to just call it a day (you can blame it on the "lab gremlins") and get the data from someone else. As frustrating as it may be to "give up" it's much more frustrating to keep repeating assays that just aren't cooperating.

N6-methyladenosine (also known as 6-methylaminopurine 9-ribofuranoside) is tested as an inhibitor of the adenosine deaminase reaction. The manner of inhibition (competitive, noncompetitive, or uncompetitive) with respect to adenosine and the inhibition constant (Ki) are determined. The effect of an inhibitor is measured over a range of non-saturating levels of substrate. The substrate is varied from 0.625 to 5 times its Km while constant levels of inhibitor are present in the assay solution. Run assays at five different concentrations of adenosine within this range then repeat in the presence of 2 different levels of inhibitor.

Make 10 to 20 ml of a solution of adenosine at approximately 30 times the Km determined in Part 2. Then make the following dilutions in phosphate buffer:

Instead of adding different amounts of the adenosine solution to the assay solution as in Part 2, the inhibition studies will be done by adding the same volume (0.5 ml) of different concentrations. Either method of substrate addition is suitable for kinetic measurements but slightly more reproducible results are obtained by adding a constant volume.

Prepare 3.0 ml assay solutions from 0.5 ml of adenosine solution plus appropriate volumes of enzyme and phosphate buffer. Obtain reaction velocities for all 5 concentrations of adenosine.
[NOTE: These results will be used to determine the Km and Km-observed (Kmobs) / Km-apparent (Kmapp) of the substrate for the inhibition studies. Do the Km and Vm compare to those determined in Part 2? Does it matter if the Vm's are the same as previously determined?]

Determine the level of N6-methyladenosine that causes 10 to 20% inhibition of the reaction rate (in nmol/min) of your most concentrated adenosine assay. This determination is accomplished by preparing an assay using the 1:1 dilution of adenosine and adding various volumes of inhibitor solution. After finding the appropriate level of inhibitor, include the inhibitor in the assay solution and measure the enzyme activity using each of the adenosine solutions. Keep the assay solution volume at 3.0 ml by changing the amount of phosphate buffer if necessary. Repeat the inhibition study using twice that amount of inhibitor.

Results

• [Draw this graph during lab]
  With the data from Part 2, construct a graph of velocity (nmol/min) versus [substrate] (in µM) to demonstrate that a saturating level of substrate has been reached. Draw a smooth curve described by the data points. Estimate Vm and Km from this plot. The Vm is the asymptote that is being approached and is read from the y-axis. Extend a horizontal line at the half maximal velocity (Vm/2) and determine the concentration of substrate where the horizontal line intersects your plot.
  NOTE: You must obtain the Km value for adenosine before proceeding to Part 3.
  On the same graph,plot the data of 2'-deoxyadenosine and determine a Km and Vm for this substrate. Compare the two parameters of these substrates and explain any differences or similarities.
  
  
• [Draw this graph outside of lab]
  From the determinations of Part 3 prepare a graph of 1/velocity versus 1/[adenosine] and plot the uninhibited velocity and the inhibited velocities on the same graph. Draw the line by hand and remember that the least accurate points will be to the far right side of the graph. A least squares fit to the inverted data will be no better and probably less accurate than the line that you draw by hand. Clearly label the line resulting from each data set. Report the type of inhibition. Determine a Ki for N6-methyladenosine from the x-intercept of the inhibited line or from the slope and y-intercept. (Consult a general biochemistry text for the equations.)
  
  *Compare the Vm's and Km's for adenosine determined in Part 2 and in Part 3.
  
  Suggested table for your notebook:

  Assay	[adenosine] in assay (µM)	1/[adenosine] (1/µM)	DA/min	velocity (nmol/min)	1/velocity (min/nmol)
  1	#	#	#	#	#
  2	#	#	#	#	#
  3	#	#	#	#	#

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 16 June 1999
Updated 11 April 2013