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Day 1: Biological Buffers

Assignments Due

Preparation

Overview of Experiment

Retention of the activity of a protein or other bioactive molecule is usually desired during the purification process. This requirement necessitates that conditions be found to stabilize the activity throughout the purification scheme. Each team of students will search the literature for buffer conditions that have been used during the purification of adenosine deaminase. Consider the possible effects of pH and other buffer additives on the retention of activity of adenosine deaminase (ADA) as you design your buffer.  Anion exchange buffer (1X buffer without additional stability components) will be prepared and stored until lab day 3.  Refer to Scopes for a discussion of buffer preparation and for parameters that stabilize enzymes.

Special Note: Mechanical pipetting devices are relatively easy to use, but practice and adherence to proper technique are required to obtain the accuracy and precision needed for quantitative measurements. Proper care is also necessary to extend the life of these expensive instruments. Proper technique is required to obtain reliable and consistent results--make sure that you are familiar with proper technique for reproducible pipetting.

Procedures

Anion Exchange Buffer Preparation

One method of making different solutions in the same pH buffer is to make the exact volume of a stock buffer solution then add the various components to aliquots of that stock. However, dilution of the buffer solution occurs with each added component. In some cases the error is negligible but significant error can result from the addition of glycerol, glucose, and salt.  A more precise method, which is highly recommended, is to make a stock of 2 times (2X) the desired buffer concentration at the correct pH. For example, to 50 ml of the 2X solution, add other components and then add water to achieve a final volume of 100 ml. It is always wise to check the pH of the final solutions with pH paper. Since you will be preparing a single buffer and will not add components for stability until AFTER anion exchange chromatography, you will prepare a 1X stock.

The selection of a specific buffer for particular pH's should be based on pKa's of the buffer component. A table of these values can be found on p. 243 (2nd ed.) or Appendix C (3rd ed.) in Scopes.

Make the solutions and determine the pH with pH paper or with the pH electrode and meter. The accuracy of a pH paper reading is in the range of ±0.2 pH units. The meter is accurate to ± 0.1 unit. Read the cautions below in the pH meter instructions. Some buffer components are harmful to the electrode and need to be added after the pH of the solution has been measured (e.g., mercaptoethanol, dithiothreitol, glycerol, PMSF). Components that may contribute to the pH, like EDTA, should be added prior to pH adjustment, or the pH needs to be checked after the addition.

As a team (3-4 students), design a buffer to use for ion exchange chromatography on the Bio-Rad Q cartridge on lab day 4. Determine the buffer composition and pH based on theoretical aspects of ion exchange and on the results of the literature search on purification of adenosine deaminase.

Record all calculations and procedures in your notebook.

To prepare the 1X buffer stock, you may need to make a dilution from a stock solution.  A useful equation is the MOLARITY DILUTION FORMULA:

M1V1 = M2V2
(where M is molarity; V is volume; 1 is initial/starting; 2 is desired/final)

If you need to weigh out a solid compound, obtain the formula weight from the container to determine how much you should add:

(g/mol) x (desired molarity) x (final volume in liters) = g


NOTE: Solutions (1 N) of the following are located near the pH meters for titration:
HCl
NaOH
CAUTION: Eye protection must be worn while handling these solutions. Keep these solutions in the designated area of the pH meters.


How to choose the buffer

Common Additives Preparation of 1X buffer stock
  1. dissolve buffer solutes in solvent so that solution volume is 10-20% LESS than the desired final volume of 1000 ml
  2. add acid or base to adjust pH
  3. "q.s. the volume" = bring the solution to desired FINAL volume (from Latin, quantum sufficit, "as much as suffices")
  4. add RO water to desired final volume
  5. confirm pH (use indicator paper)
  6. add other components to final purified protein (on lab day 4)

Determination of pH

pH indicator strips

Paper and strips for pH determinations are selected for the desired range and accuracy. Broad range indicators yield estimates of ±0.5 pH units. Narrow range indicators reduce the error to ±0.2 pH units. The advantages of these indicators are speed, portability, relative insensitivity to additives in the solution, and small volume of sample needed. No calibration is required, and they can be used through the normal range of temperatures used in protein laboratories.

Suggestion: Never dip the strips into your buffer. Instead, remove a drop of the solution with a transfer pipette or glass rod and place it on the indicator strip. This precaution prevents your solution from being contaminated by compounds that may leach from the strip. This also allows several readings from a single length of indicator paper.

pH meters

Electrodes and meters designed to measure pH can be more accurate than the paper indicators but require more time for the readings and calibration. Follow the instructions for proper use of pH electrodes.

Caution: The pH electrodes are fragile and easily rendered useless. The electrode can be permanently damaged if placed in solutions that cause a precipitate with ions of potassium or silver or solutions that can clog very small pores. Common solution components that should not be placed in contact with a single junction electrode include: Tris, DTT (dithiothreitol), Beta-mercaptoethanol, SDS, protein and DNA. Indicator strips should be used when these agents are present. The NEWER electrodes are double junction and can be used to adjust the pH of Tris and other solutions that can damage single junction electrodes.

***The small glass bulb located at the end of the electrode is quite fragile. Do not contact the bulb with stir bars or edges of beakers.***

Do not dry the electrode with toweling. A static charge, which will interfere with the readings, can be induced on the electrode by wiping with a KimWipe. Excess liquid can be carefully blotted from the electrode but avoid excessive wiping.

Calibration of the pH meter

The calibration of the pH meter uses two calibrated buffer solutions to establish a fit to the Nernst equation. The "Standard" is the pH 7.0 solution and either pH 10.0 or 4.0 solutions are used to determine the slope.

Use of the pH Meter

Instructions for calibration with manual temperature compensation in the pH/AUTOLOCK mode (Instructions adapted from the instruction manual by JENCO Instruments, Inc., pp. 7-9)

  1. Rinse the pH electrode in distilled water and immerse in buffer 7.00. The standard solution must be stirring and the immersion level of the probe should be the same as you will use for the sample solution.
  2. (Optional step) Set the instrument to display the temperature of the buffer. Read the ambient temperature from the thermometer suspended near the pH meters and use this as the buffer temperature of the standards. Set the temperature by pressing the DIGIT key and increment the blinking place value using the COUNT key. Pressing the DIGIT key again will activate the next place value which can be advanced using the COUNT key. Limits of the instrument require that the temperature be set to less than 60.4°C.
  3. Press the STAND key. The STAND annunciator will stop flashing and the WAIT annunciator will flash, indicating that the instrument is waiting for a stable reading. The display will be locked to the buffer value corresponding to the temperature of the buffer 7.00 as set in step 2. When a stable reading is obtained, the WAIT annunciator will turn off. The SLOPE annunciator will start to flash, indicating that standardization at buffer 7.00 is complete and the instrument is ready for the second buffer to be used to determine the slope of the Nernst equation. The choice of the second buffer (pH 4.01 or 10.01) is dependent on the direction of your target pH from the standard.
  4. Remove the electrode from the reference solution, rinse well with distilled water, and immerse in the second reference buffer. (Optional:) If the temperature of the second buffer is different than the first reset the temperature as in step 2.
  5. With the reference solution stirring, press the SLOPE key. The slope annuciator will stop flashing and stay on. The WAIT annunciator will start to flash, indicating that the instrument is waiting for a stable reading. The display will be locked to the second buffer value corresponding to the temperature of the second buffer as set in above. When a stable reading is reached the WAIT annunciator will stop flashing and stay off. The instrument is now dual point calibrated and is ready for measurements within the range of the standards used.
  6. Rinse the electrode and suspend it in your buffer solution. To obtain a reading of a solution the "autolock" feature is convenient because the reading "locks" on the first stable pH reading obtained. However, this feature becomes a nuisance when one is trying to adjust the pH of the buffer to a desired pH. Disarm the "autolock" feature by pressing the "MODE" button until "pH" replaces "AUTOLOCK" in the bottom left corner of the display. This allows continuous reading of the pH necessary for titrations.
ERROR messages
Er1
pH electrode offset greater than ±1 pH unit. Electrode not functioning or reference buffer is not 7.00.
Er2
pH electrode sensitivity off by more than 20%. Electrode not functioning or buffers 4.01 or 10.01 are not correct.
Er3
Temperature out of the 0 to 100°C range.
Er4
Buffer temperature out of the 0 to 60.4°C range.
Er5
pH values out of the -2.0 to 16.00 range or mV values out of the -999 to +999 range.
Er6
Illegal operation procedures.

Spectrophotometric Quantitative Assay for Adenosine Deaminase

*Record all instrument settings and data in your notebook. (This assay will be used throughout the course.)
Gather reagents and supplies at the spectrophotometer. A wash bottle with RO water should be used to rinse the cuvettes and a large beaker can be used as a waste reservoir.


ATTENTION: there are 3 different models of spectrophotometers; please follow the appropriate instructions for the spectrophotometer you choose

1) Genesys 5 spectrophotometer

2) Biowave S2100 UV/Vis Diode Array spectrophotometer

3) Libra S22 spectrophotometer

Sample preparation

Obtain an aliquot (30 µl) of concentrated adenosine deaminase. Remember to keep the enzyme solutions at 4°C ("on ice") at all times.  Prepare the following samples: make 100 fold dilutions (i.e., 1 part ADA in 100 parts TOTAL, 1:100) of the enzyme into 50 mM KPO4 (pH 7.4) [one sample prepared by each team member] (the phosphate assay buffer has been prepared for you); mix thoroughly by inverting or "flicking" several times. Any reasonable volumes may be used for these dilutions, although do not exceed 0.5 ml for the final volume or you will not have enough stock enzyme. Microcentrifuge tubes (1.5 ml) can be used for easy handling and storage of these volumes.The stock solution of enzyme is dense and tends to sink to the bottom of the tube.

    ADA Assay

  1. Prepare a quartz cuvette containing ADA assay buffer (3 ml 50 mM KPO4, pH 7.4).


  2. Add 0.03 ml of 10 mM adenosine (the substrate) to the cuvette containing ADA assay buffer.

  3. Hold a layer of Parafilm over the top of the cuvette with your finger and mix by inverting several times. Carefully clean the clear sides of the cuvette with a KimWipe. One square of Parafilm can be used for several assays--just move to a clean area of the film each time.

  4. Do a BLANK RUN: [NOTE: you only need to perform the "BLANK RUN" once.]


  5. When the blank run is complete, add 20 µl of the enzyme solution (ADA diluted 1:100 in assay buffer) to the cuvette containing the reaction solution described above, cover the cuvette top with Parafilm, and invert gently 3-5 times. (Never shake a protein solution as foaming may occur and denature the protein.) Clean the exterior of the cuvette carefully with a KimWipe and place the cuvette in the spec and initiate another measurement.


    At the end of the run observe the graph for linearity and check that the calculated rate of the enzyme solution is between a rate of -0.02 to -0.06 A/min. Adjust the volume of control enzyme solution added to make the rate fall in the given range and repeat a reaction measurement if necessary. NOTE: if you need to repeat a measurement or use a different volume of enzyme, you must prepare a FRESH reaction cuvette (i.e., fresh assay buffer and substrate).

  6. Prepare a fresh reaction solution (i.e., assay buffer, adenosine, and diluted enzyme) containing a constant amount of solution (i.e., the SAME volume as used above) and measure the rate of reaction. Repeat this assay for a total of three assays per team member.  (You do not need to do a BLANK RUN for these reactions.)


    Between runs, rinse the cuvette with RO water at least 3 times and invert on a KimWipe to drain. Two cuvettes are available at each station: while one is being measured, the other can be cleaned and prepared with assay buffer and adenosine for the next run; initiate the reaction with the addition of the enzyme just prior to the measurement.

    A residual layer of liquid does not interfere with subsequent readings. Do not dry the inside of the cuvette with a tissue as this leaves small fibers that interfere with the absorbance readings.
Additional Notes:
  1. Record the rate in ΔA/min and indicate whether the graph appeared linear (record the correlation coefficient (Libra S22 or Biowave instruments) or the linearity (Libra instrument) if applicable). Make sure you record the assay results for each member of your team.
  2. Calculate the average rate (use the absolute value of ΔA/min) +/- standard deviation for each member of the team.  How consistent were you with your assays? What factors may have contributed to variability?  How might variability affect the outcome of future experiments?  What are some things you could do to minimize this variability?

    NOTE: For the protein purification table and the kinetic study, conversion to µmol/min or nmol/min is required. The extinction coefficients for these conversions should be found by someone in the library assignment.

 


 

Copyright, Acknowledgements, and Intended Use
Created by B. Beason (bbeason@rice.edu), Rice University, 25 May 2010
Updated 1 May 2013