BioChem Procedures S15

7/25/2019 BioChem Procedures S15

1/61

BIOCHEMISTRY LABORATORY

RULES AND PROCEDURES

It is essential for all concerned that certain rules be followed while in the laboratory. Read the

following carefully and ask questions necessary for clarity before signing the statement.

1. Goggles will be worn at all times when performing experimentsor when others are performingexperiments. You will be allowed two verbal warnings. After this, a grade penalty will result.

2. Full shoes are recommended. Sandals and flip-flops are an accident waiting to happen.

3. A lab coat or apron is required when wearing shorts, or other clothes which expose skin.

4. Attendance is required at all laboratory sessions. To skip a session due to your being ahead, prior

permission is required.

5. Do not use flames unless specifically instructed to do so. Do not leave flames unattended. Turnburners off when not in use.

6. Remember that most organic chemicals are flammable, toxic, carcinogenic or all three. Treat them

accordingly.

7. You should acquaint yourself with the eyewash station, safety shower and fire-fighting equipment,including their location. Dont be surprised if this is a test question. Label all containers.

8. No smoking, eating or drinking is allowed in the lab. If you are taking a prescription or other drug that

will affect alertness, notify the instructor before going into lab.

9. No students are allowed in the prep area without the lab instructor. No students are allowed in Dr.

Boles or Dr. Jiang research labs without permission.

10. Report all accidents or injury to the instructor immediately.

11. Dispose of organic chemicals only in designated containers, unless directed otherwise by the lab

instructor. Be sure waste containers remain closed and are appropriately labeled.

12. You are financially responsible for all glassware and equipment issued or checked out from thestockroom. Clean your area before leaving the lab each day.

13. Keep your notebooks in your drawer unless instructed to take them home. Notebook grading will

sometimes not be announced.

I have received a copy of the above rules and agree to abide by them. I understand the above rules andhave had any questions explained. I agree to accept appropriate corrective measures if violations are

incurred.

Name (Print) _____________________________ Signed __________________________

Date ____________________


2/61

GUIDELINES FOR WORKING IN BIOCHEM LAB

1. Bring a pair of goggles. Keep a roll of paper towels in your drawer.

2. Use of balances:

Never set an object or material to be weighed directly on a balance pan. Always use a piece ofpaper as a tare (so as not to deface the balance pan) and use the automatic tare device.

Use a forceps (not your fingers) to pick up glass, metal and porcelain objects to be weighed isrecommended, but not always required.

The loss of heat from these objects (transmitted to them from your fingers) will result influctuations in readings on the analytical balance.

Record analytical balance readings to four (4) decimal places unless instructed otherwise.

When finished using an analytical balance: Leave balance and the area around the balance

clean. Shut both doors of balance.

3. Clean your equipment and rinse with distilled water before leaving the lab each period. Youwill need glassware clean and dryfor subsequent experiments.

4. Cleanyour bench space before you leave. Put ring stands on the floor against the wall.

5. Each person is responsible for keeping a lab notebook (no three-ring binders) even though

most experiments will be performed in groups.

GUIDELINES FOR LABORATORY NOTEBOOKS

1. Leave the first few pages of the notebook blank to be used for a Table of Contents

2. Number each page of the notebook and add new experiments to the Table of Contents, by Title

with page numbers, as they accumulate.

3. Each page is to be dated at the top and signed at the bottom as data is entered.

4. Record all data directly into the notebook. Do not transfer data from scraps of paper, etc.The notebook is an active record of data collected, carry it to the balance room, etc.Describethe experimental steps in details in your notebook.

5. Make entries in ink only. Never completely scratch out data. Draw a single line througherrors. NEVER ERASE OR USE WHITE OUT!!!

6. Never record data without identification. Be sure to use correct units with all data. Show

buret readings, not just net volume and make all buret readings to two decimal places.


3/61

GUIDELINES FOR LABORATORY NOTEBOOKS - continued

7. Make all calculations in your notebook. (Show the set up for the calculation if more thanone calculation of the same type must be made).

8. Any printouts (including graphs) that are taped into the notebook should be treated as follows:Sign your name across the graph, tape and notebook in at least one place.

9. Plots or graphs made with Excel, etc., should be titled and labeled similar to the followingexample:

REACTION RATE vs CONCENTRATION

Reaction

Rate (units)

TITLE: The title should indicate what the graph is for. The title should be informative.

AXES: Label both ordinate and abscissa. Include units and numerical values along each axis.

A line through the data may not necessarily touch each data point. This is because of theexperimental error incurred in the determination of each piece of data. If there is little

experimental error, then the data points will be very close to the line through the data, however, ifthe experimental error is great, then none of the points may be on the best line through data points.

Draw a smooth line (or curve) through data points which appears to best fit the relationshipindicated by the points (linear, curved, sigmoid, etc.).

10. Use subheadingsoften in your notebook to identify 1) what you were doing and 2) the data

that was collected for a particular part of the experiment. Underline Headings and Subheadings.

11. Your conclusions at the end of each experiment should discuss observations concerning yourdata. This should not simply be a reiteration of your data, but rather a discussion.


4/61

12. Label your tubes with your name and the description of the content.

13. Record what happens during the experiment instead of whats in the handout. For example, ifthe handout says the temperature is around 95

oC and the temperature you use is 97

oC. Please

record in your notebook that you are using 97oC .

14. For all the instruments you use for the experiment, you need to record the manufacture, themodel number, the parameters and the software you use. If the instrument is not in thebiochemistry lab room, please specify where the instrument is located.

15. Pay attention to the decimal.

17. If you made a mistake during the experiment, please record honestly. You are not going to getpoints off if you correct the mistake. But if the instructor notices your mistake and you didntrecord, the instructor will take points off when grading.

18.Calculation, you need to show your work step by step to let the other people who read yournotebook to understand.


5/61

PREPARATION OF BUFFERS

A number of phosphate buffers will be prepared during this lab period using the concepts and

calculations presented in CHEM 4610. For H3PO4 use pK1 = 2.3; pK2 = 7.2 ; and pK3 = 12.3.

Always use distilled water to prepare solutions.

PART 1:

Prepare 100 ml of 0.05 M phosphate buffer, pH 6.3, using solid NaH2PO4and solid Na2HPO4.(See the containers for actual formulas and molecular weights.)

Calculate the amounts of the two solids needed for this buffer.Weigh the solids on the analytical balance and transfer to a 100 ml volumetric flask.

Fill the flask about 2/3 full with water, dissolve all the solid then fill to the mark and mix well.Measure and record the pH of this buffer with MEASURENET.

PART 2:

Prepare 100 ml of 0.05 M phosphate buffer, pH 6.3, using 0.05 M NaH2PO4and0.05 M Na2HPO4.

Calculate the volume of each of the two solutions needed for this buffer.Combine these volumes in a beaker using a 100 ml graduated cylinder as a measuring device.

Mix well.Measure and record the pH of this buffer with MEASURENET.

NOTE FOR PARTS 3 & 4,I:

The source of the buffers to be prepared in these parts will be 2 M phosphoric acid.The proper conj. acid/conj. base ratio will be obtained by adjusting with 1 N NaOH.

Use a 5 ml Mohr pipet to measure the volume of 2 M H3PO4needed.Use a 50 ml buret to measure the volume of 1 N NaOH needed.

PART 3:

Prepare 250 ml of 0.02 M phosphate buffer in which [H2PO4]- = [HPO4]

=.

Calculate the volumes of 2 M phosphoric acid and 1 M NaOH required.To a 250 ml volumetric flask containing about 100 ml of water, transfer the required volumes

of 2 M phosphoric acid and 1 M NaOH, then fill to the mark with water and mix well.Measure and record the pH of the buffer with MEASURENET.


6/61

PART 4:

I. Prepare 250 ml of 0.05 M phosphate buffer, pH 7.4, using 2 M H3PO4and 1 N NaOH.Use a 5 ml Mohr pipet to measure the volume of 2 M H3PO4needed.

Use a 50 ml buret to measure the volume of 1 N NaOH needed.

To a 250 ml volumetric flask containing about 100 ml of water, transfer the required volumesof 2 M phosphoric acid and 1 M NaOH, then fill to the mark with water and mix well.Measure and record the pH of this buffer with MEASURENET. Keep this buffer for (II), below.

II. Transfer 100.00 ml of the buffer prepared in (I) above into a 150 ml beaker using a buret.

First, clean the buret. This is not as easy as it sounds because NaOH adheres to glass fairly well.So, proceed by rinsing the buret with water followed by a rinse with a little dilute HCl (run the

HCl through the buret tip during this process). This neutralizes any residual NaOH and theresulting salts and HCl can then easily be rinsed out with water. After this has been done, rinse

the buret with some of the buffer from (I), drain the buret well then fill it with the buffer andyou are ready to dispense the buffer.

To this 100.00 ml of buffer, add 1000 l of 2.00 N HCl. Mix well.

Assume no volume change and calculate the new pH of the buffer.Measure and record the pH of this resulting buffer solution with MEASURENET.


7/61

EXAMPLE: ENTER THE ITEMS BELOW INTO YOUR NOTEBOOK:Show your calculations and be sure to subtitle your work as needed, i.e., Preparation of a 100

mL 0.05 M Phosphate buffer, pH 6.3

1. mmole of total phosphate needed in buffer (show all calculations in each section)

mmol of [H2PO4]

-

neededweight of [H2PO4]- needed

mmole of [HPO4]=needed

weight of [HPO4]=needed

Measured pH

2. mmole of total phosphate needed in buffer

mmol of [H2PO4]- needed

ml of 0.05 M [H2PO4]-needed


ml of 0.05 M [HPO4]=

needed

Measured pH

3. mmole of total phosphate needed in buffervolume of 2 M H3PO4needed

mmole of NaOH needed to get [H2PO4]-= [HPO4]

=

volume of 1 N NaOH needed

Predicted pHMeasured pH

4.I. mmole of total phosphate needed in buffer

volume of 2 M H3PO4neededmmole of NaOH needed to get to pH 7.4

volume of 1 N NaOH neededMeasured pH

4.II. mmole H2PO4-in 100 ml of buffer I

mmole HPO4=in 100 ml of buffer I

mmole HCl added

mmole H2PO4-after addition of HCl

mmole HPO4=after addition of HCl

Calculated pHMeasured pH

Include observations/discussions about your results in your notebook.

The following tables are provided for you to use as scratch paper, for pre-lab calculations, etc., or

however you see fit to use them. Be sure to be very descriptive in your notebooks as to how thesebuffers are prepared. If you choose not to use mmoles, be sure your calculations are clear.


8/61

PH/BUFFER WORKSHEET NAME ___________________

PART 1 0.05M

mmole of total phosphateneeded in buffer

mmol of [H2PO4]- needed

weight of [H2PO4]- needed


weight of [HPO4]=needed

Measured pH

PART 2 0.05M


mmole of [H2PO4]- needed

ml of 0.05 M [H2PO4]-

needed


ml of 0.05 M [HPO4]=

needed

Measured pH


9/61

PART 3 0.02 M


volume of 2 M H3PO4

needed

mmole of NaOH needed to

get [H2PO4]-= [HPO4]

=

volume of 1 N NaOH

needed

Predicted pH

Measured pH


10/61

PH/BUFFER WORKSHEET NAME ___________________

PART 4, I 0.05 M

mmole of total phosphate

needed in buffer

volume of 2 M H3PO4

needed

mmole of NaOH needed toget to pH 7.4

volume of 1 N NaOHneeded

Measured pH


11/61

PART 4, II

mmole H2PO4-in 100 ml of

buffer I

mmole HPO4=in 100 ml of

buffer I

mmole of HCl added

mmole H2PO4-after

addition of HCl

mmole HPO4=after addition

of HCl

Calculated pH

Measured pH


12/61

TITRATION OF AN AMINO ACID

In Part I of this experiment, the amino acid glycine will be titrated as well as a water blank.

The pKa value of the basic functional group of GLY, pKa2, will then be determined graphically.

In Part II, the acid end of the titration curve will be plotted from calculations using the

Henderson-Hasselbalch equation and pKa1of glycine.

PART I TITRATION OF AN AMINO ACID

SAMPLE PREPARATION:

Weigh out approximately 400 mg of glycine on an analytical balance. Record this mass.Transfer to a 150 ml beaker and dissolve in 50.0 ml of distilled water. Titrate this sample as

indicated below using 1.0 N NaOH. Record and use the exact normality as written on the bottle.

TITRATION:

There is one pH meter available per group. Before beginning a titration, record the initial pHof the solution. Add base dropwise and after each addition record the pH and the buret reading.

Mix well by swirling after each addition or using a magnetic stirrer. The amount of base requiredto produce a pH change will vary during the course of the experiment, add enough drops to get an

observable change. Stop the titration when a pH of approximately 12 is reached. Record data inyour notebook.

When finished with the amino acid titration, titrate a water blank, i.e. titrate an amount of

water equal to that used to dissolve the amino acid. Again, use 1.0 N NaOH as the titrant.Conduct this titration in the same manner as the previous one. Record data in your notebook.

DATA WORKSHEET AND PLOTS:

Plot the data from the base titrations of the amino acid and water on the same graph. If you use

graph paper rather than Microsoft excel, use graph paper which has 10 divisions/cm and use astraight edge and/or french curve to make smooth lines. Plot pH on the ordinate and titrant on the

abcissa. The best smooth line will follow the experimental points fairly well. This is Plot I. Youmay use excel, but do not allow excel to interpolate your results. You may need to use a French

curve if you are not good at free-hand. Tape the plot into your notebook and include all datatables.

Tabulate data from above curves in increments of 0.2 pH unit starting at the initial pH and

ending at pH 12. These data are to be read from the smooth curves (Plot I) which were drawnfrom the original titration data. Because a smooth curve will not necessarily go through all of the


13/61

experimental points the tabulated data will be differentfrom the experimental data. Record datain your notebook and label accordingly.

The net volume is the numerical difference between the data from each Plot I curve at a given

pH. The net volume represents the volume of base required to titrate the amino acid itself without

contribution from the solvent and should be recorded in your notebook.

Finally, plot pH vs the net volume readings to obtain a corrected titration curve (Plot II). This

second plot of pH vs net mL will have the ends parallel to the pH axis due to the fact that the endsof the amino acid titration curve and the water titration curve in Plot I are parallel to each other.

Leave room on the bottom half of the paper (if not using Excel) to plot the acid end of the titrationcurve to be determined in Part II of this experiment.

GRAPHICAL DETERMINATION OF pKa2

Draw parallel lines 1 & 2 along the vertical parts of the titration curve (Plot II). Draw line 3

parallel to lines 1 and 2 and half-way between 1 and 2. The pH corresponding to the intersectionof line 3 with the titration curve is pKa2.

PART II CALCULATION OF A TITRATION CURVE

CALCULATION:

Use the Henderson-Hasselbalch equation and a pKa1of 2.30 for glycine to determine the acid

portion of the titration curve. Assume an amount of glycine equal to that used in the base titration(Part I).

Assume that all of the glycine is initially in the completely protonated form and make calculationsrepresentative of every 0.2 ml of NaOH added from 0.2 ml to 5.2 ml. Also, make a calculation

for 5.3 ml of base added. These values must be adjusted based upon the mass of Glycine and theconcentration of base.

PLOT:

Plot these data (pH vs ml of base added) on Plot II with the tabulated experimental data. You

may recreate Plot II from your excel file and include it if you wish to use excel. Connect the endsof the titration curves in the neutral region. This yields a complete titration curve for glycine.


14/61

Include in your notebook:

1. The value of pI determined from Plot II , also indicate pI on Plot II

2. Calculate the pI from the pKa's as given in the text.

3. Define equivalent weight.

4. What is the equivalent weight of glycine ?

5. Indicate pK2on Plot II


15/61

PART I:

EXAMPLE FOR NOTEBOOKORIGINAL TITRATION DATA GROUP_____________________

AMINO ACID WATER

pH Vol (mL) pH Vol (mL) pH Vol (mL) pH Vol (mL)

EXAMPLE FOR NOTEBOOK

TABULATED DATA

pH AA plot Water Plot Net Vol (mL)

PART II: Format your own data entry for the notebook.


16/61

Homework (sample quiz preparation). Rewrite these questions in your notebook with

appropriate answers.

+NH3|

Using the general formula R - CH - COO-for an amino acid, draw the structure(s) which would be

present:

1. In strong base: 2. In strong acid:

3. At the pI: 4. At pK1:

5. At pK2:

.


17/61

SPECTROPHOTOMETRIC DETERMINATION OF TRYPHTOPHAN AND

TYROSINE IN CHYMOTRYPSINOGEN-A

References: Wetlaufer, Advances in Protein Chemistry, 17, 304 (1962).Edelhoch, H., Biochemistry, 6, 1948 (1967).

INTRODUCTION:

The standard procedure for determining the amino acid composition of proteins involves acidhydrolysis. Because tryptophan is destroyed during acid hydrolysis another method must be usedfor its determination. This spectrophotometric procedure is one of several methods currently

available.

The absorbance of a protein between 270 nm and 300 nm may be attributed essentially to itstryptophan and tyrosine content (see plot of absorbance vs. wavelength in lab). Theoretically, itshould be possible to estimate the tryptophan and tyrosine content of a protein from its ultraviolet(uv) spectrum. In doing so, three practical problems are encountered.

(1) The molar absorptivity (extinction coefficient) of a tryptophan or a tyrosine residue ina protein depends upon its local environment. In a folded protein molecule, tryptophan andtyrosine residues on the surface of the molecule will have different absorptivities than those on theinside which are shielded from the solvent. The problem is overcome by unfolding the protein(dissolving it in 6 M guanidine- HCl) and thereby exposing all of the tryptophan and tyrosineresidues to the solvent.

(2) The molar absorptivities(!) of tryptophan and tyrosine residues in a protein differ fromthat for the free amino acids. This problem is minimized by using the values for N-acetyl-L-tryptophanamide and glycyl-L-tyrosylglycine which are more representative of the amino acidresidues.

(3) Even when using these reference compounds the absorbance spectrum of a protein in6 M guanidine-HCl is not identical to an equi-residue concentration of the reference compounds in6 M guanidine-HCl. This is due to a small contribution which cystine residuesmake to theabsorbance and the fact that the protein may not be completely unfolded. By correcting for theabsorbance of cystine and by measuring the absorbance of the protein at wavelengths where thereis the least difference from the model compounds, acceptable results can be obtained provided thatother aromatic groups (coenzymes or impurities) are not present.


18/61

Edelhoch suggests that the tryptophan and tyrosine content of proteins be determined from thefollowing equations.

!280= 5690 W + 1280 Y

!288= 4815 W + 385 Y

The contribution of tryptophan and tyrosine residues to the molar absorptivity of the protein in 6M guanidine - HCl at 280 nm and 288 nm are represented by !-280 and !-288, respectively. Thevalues of W and Y are the numbers of tryptophan and tyrosine, respectively, that occur in theprotein.

PROCEDURE:

The ultraviolet spectrum from 270 and 300 nm will be obtained using an automatic recordingspectrophotometer. A chymotrypsinogen-A stock solution has been prepared which contains

approximately 14 mg/ml of zymogen in 0.001 M HCl.

Part I: Determine the Concentration of Chymotrypsinogen-A

1. Pipet 3 ml of phosphate buffer into the two matched (plastic) uv cells and place them in thefirst two compartments of the spectrophotometer. The blank should be in the firstcompartment and the sample should be in the second compartment. You will beinstructed in the proper use of the uv/vis spectrophotometer.

2. Zero the blank at 280 nm (the front cell). Measure the absorbance of the sample cell. Thisslight absorbance you read represents the difference between the two cells. If it is positiveit should be subtracted from the subsequent reading and if it is negative it should be added.Be sure to note the position of the uv cells in the spectrophotometer. Each time the cellsare placed in the instrument their orientation should be the same or this difference willchange and error will be introduced. The side facing the front of the instrument should bemarked with a marker or by some other means.

3. Remove the sample cells from the spectrophotometer. Pipet 0.100 ml of thechymotrypsinogen-A stock solution into the sample cell using an Eppendorf pipet. Place asmall piece of parafilm over the cell and invert two or three times to mix (DO NOTSHAKE). Also, add 0.100 ml of 0.001 M HCl to the reference cell, mix, then replace thecells in the spectrophotometer and read the absorbance of the sample vs. the phosphatebuffer blank at 280 nm.

4. Calculate the molar concentration of the protein from the relationship: Abs(280) = 2.0 xconc. in mg/ml......note: the 2.0 is referred to as an optical factor that in part takes intoaccount the extinction coefficient of the protein. Assume that the molecular weight ofchymotrypsinogen-A = 25,000 g/mole.


19/61

Part II: Record the Spectrum of the Denatured Zymogen

1. Prepare 10 ml of 6.2 M guanidine-HCl by placing an appropriate amount of solidguanidine-HCl into a 10 ml volumetric flask (provided). This requires some

patience...make sure that ALL of the solid is in the flask before adding ANY buffer.2. Dissolve and bring up to volume with 0.02 M phosphate buffer (provided).3. Pipet three ml of the 6.2 M guanidine-HCl into each of the two uv cells using a pipet

(provided).4.

Pipet 0.100 ml of the 0.001 M HCl into the reference cell (blank), and pipet 0.100 ml ofthe chymotrypsinogen-A stock soluton into the sample cell.

5. Invert to mix as before then allow the solutions to stand for 30 minutes while theguanidine-HCl interacts with the zymogen.

6. Record the spectrum of the sample vs. the blank. Read the absorbance at both 280nm and288 nm. You will be instructed in the operation of the uv/vis spectrophotometer.


20/61

CALCULATIONS:

One can obtain the absorbance value at any given wavelength by using the cursor in thescanning software. Determine the absorbance at 280 nm and 288 nm of the zymogen inguanidine-HCl.

Next, calculate the contribution of cystine to the absorbance of the protein at 280 nm and 288nm in 6 M guanidine-HCl from the Beer's Law relationship and the concentration of the protein asdetermined in Part I. Assume that there are 5 cystines in chymotrypsinogen-A and that the molarabsorptivities of cystine in 6 M guanidine-HCl are 120 cm2/mmol and 70 cm2/mmol at 280 nmand 288 nm, respectively.

The net absorbance due to the sum of tryptophan and tyrosine is found by subtracting thecalculated absorbance due to cystine from the total absorbance of the protein determined in Part II.Using this net absorbance, calculate the molar absorptivity due to the sum of tryptophan andtyrosine at each wavelength and use Edelhoch's equations to determine the number of tryptophans

and tryosines in the protein.

The cells (cuvets) used in a uv-visible spectrophotometer can be made of glass, plastic orquartz. The wavelengths of interest in this experiment, 270 - 300 nm, are in the uv region andquartz cells are usually used due to interference exhibited by some glass and plastic in the uv.However, quartz cells are expensive (about $200/pair) and there are now available some excellentacrylic cells which work well in the uv (we will use these). Remember, fingerprints absorb in theuv so be sure to wipe off the optical surface of the cells with a kimwipe prior to making ameasurement or scan. For Part II of the procedure, clean the cells from Part I or use a second setof clean cells.


21/61

Additional data to be included in the Results section of your notebook for this experiment:

Reproduce these questions with the data and calculations that are required.

1. Calculate the amount of solid guanidine-HCl needed to prepare 10 ml of 6.2 M solution.

2. Weight (from toploading balance). ____________

3. Calculate the concentration of guanidine-HCl in the cell in Part II after addition of thezymogen.

4. Net absorbance of chymotrypsinogen-A at 280 nm from Part I. __________

5. Chymotrypsinogen-A concentration.

a. mg/ml (from abs. in item 4 and the optical factor): b. Molarity, M (need M.W.):

6. Number of cystines in chymotrypsinogen-A. _______________

7. Total concentration of cystine in chymotrypsinogen-A, M (from items 5b and 6):

8. The Beer-Lambert formula is: ____________________________

9. Calculate the absorbance due to cystine.

280 nm: 288 nm:

10. Absorbance of chymotrypsinogen-A after standing in 6 M guanidine-HCl (Part II).


22/61

280 nm: __________ 288 nm: __________

11. Net absorbance due to tryptophan and tyrosine only (subtract cys-cys: Item 10 - item 9)

280 nm: __________ 288 nm: __________

12. Calculate the molar absorptivities for the sum of TRP and TYR.

280 nm: 288 nm:

13. Using Edelhoch's equations calculate the number of TRP (W) and TYR (Y) inchymotrypsinogen-A.

14. What are the actual numbers of TRP and TYR in chymotrypsinogen-A? (can be from internet)

TRP _______, TYR _______ Reference: _________________________

15. In the Beers law equation, absorbance is unitless, !is in cm2/mmol and cell path = 1 cm.Calculate units on the concentration term.


23/61

PROTEIN DETERMINATION USING COOMASSIE BRILLIANT BLUE G-250

Reference: M.M. Bradford, Anal. Biochem. 72, 248 (1976).

Bradford assay is a common used method to determine protein concentration in the lab. It ismore tolerant of interferring substances than the Lowry assay, requires less mechanical

manipulation and less time. This method will also be used to determine protein concentration. TheBradford Method is called a dye binding assay. The dye that is used binds with protein to yield a

colored complex and the intensity of this color is proportional to the amount of protein present.The dye used is Coommassie Brilliant Blue G-250.

Based on the information provided, please find the reference article and include in yournotebook.

REAGENT PREPARATION:

The Bradford reagent is somewhat stable but should replaced weekly or checked to ensurereproducibility of data. This reagent will be prepared and ready for use in lab.


24/61

PROCEDURE:

Each laboratory group will be assigned a protein for which a standard curve is to be prepared.

PROTEIN STOCK SOLUTION:

Make 50 ml of protein solution which is 2.0 mg/ml. Use 0.15 M NaCl to dissolve the proteinand bring it to volume (see below). One group may be asked to prepare this NaCl solution for the

entire lab - see instructor. Each group will prepare their own protein stock solution.

Weigh the required amount of protein and dissolve in a small amount of 0.15 M NaCl using a30 ml beaker. Stir gently with a glass rod, crushing small pieces of undissolved material, until all

dissolves. Transfer into a 50 ml volumetric flask. Quantitatively wash the beaker with 0.15 MNaCl and add the washings to the volumetric flask. Bring to mark with 0.15 M NaCl. This

solution is Protein CSand will be used to prepare protein solutions of varying concentrations foranalysis as directed in the table below.

PROTEIN SAMPLES TO BE ANALYZED:

TABLE C1

Sample tube # ml Protein CS ml 0.15 M NaCl

Blank 0 NaCl only

1 0.1 1.9

2 0.1 0.9

3 0.2 0.8

4 0.4 0.6

5 0.6 0.4

6 0.8 0.2

7 stock only 0

Calculate the concentration of protein in each solution above in microgram (g) per ml and recordin your notebook. Two determinations should be made at each concentration.


25/61

ANALYSIS PROCEDURE:

This method is best for protein samples containing 20-300 microgram of protein, however,

there is an alternative microassay that can be carried out which allows detection of as little as 1

microgram of protein. The alternative microassay incorporates a different concentration of the dye,however.

Prepare assay solutions from each of the samples in Table T1, including the blank, as follows:

1. Pipet 0.1 ml of a protein sample into a testube. Add 5 ml of Commassie Blue reagent andmix by vortex or inversion. Read absorbance at 595 nm after 5 minutes but before 1 hour.

Subtract the absorbance of the blank at 595 nm from each of the protein sample absorbances to

obtain a net absorbance for each protein sample analyzed or zero the instrument on your blank andrecord the actual absorbance of each sample.

STANDARD CURVE: Prepare a standard plot of absorbance at 595 nm vs. g analyzed.

PIPETS:

Pipet provided Use

Micropipette 0.15 M NaCl solution and1 ml protein samples from Table C1

Micropipette Protein CS

5 ml Mohr Commassie Blue reagent


26/61

RESULTS (Be sure to indicate the page(s) where raw data is recorded in your

notebook)

COOMASSIE BLUE G-250

1. Weight of protein: __________________

2. Concentration of protein in samples and Absorbance at 595 nm vs the blank: (may be

in excel)

Sample # Conc. g/ml g in 0.1 ml Abs, meas. Abs, net

Blank

1

2

3

4

5

6

7

Plot absorbance vs g protein in 0.1 ml (may use excel).

3. What are two substances that interfere with the assay? (see literature)

4. What affect does this assay have on the cuvettes? How can you clean them?


27/61

Crystallization

X-ray crystallography

X-ray crystallography is a method to determine the protein 3-D structure at atomic level.

It allows X-ray beam to strike a protein crystal frozen in liquid nitrogen and thediffraction pattern (known as reflection) are collected to reconstruct the 3D structure ofthe protein.

How to crystallize protein

Crystallization is more art than science. The principle of crystallization is to try

everything. There are three methods of preparing crystals, hanging drop, sitting drop andmicro-dialysis based on the position of protein solution and reservoir solution. In this lab,

we are going to set up crystallization of lysozyme that is a good example forcrystallization demonstration.

Reagents:

Lysozyme 100mg/ml, 40 mg/ml, 20 mg/mlLysozyme crystallization buffer: 30% w/v PEG MME 5,000, 1.0 M Sodium chloride,

0.05 M Sodium acetate trihydrate pH 4.6Lysozyme crystallization plate

Cover slideForceps

Pipette

Protocol

Use P1000 micropipette to transfer 500 l of lysozyme crystallization buffer to one cell

in the plate as reservoir solution.

Take a glass cover slide from the slide box with forceps and gently put it on the table.

Use P2 micropipette to transfer 1 l of lysozyme solution (100 mg/ml) to the glass coverslide. Be careful since 1 l is a very small amount and the glass cover slide is fragile. And

then take 1 l of reservoir solution and add into protein solution on the cover slide.


28/61

Use forceps to carefully take the cover slide with the protein drop and then slowly flip thecover slide over to put on top of the cell which you added reservoir solution.

Press gently to seal.

Repeat the same procedure for lysozyme 40 mg/ml and lysozyme 20 mg/ml.

Once everybody set up the experiment, hand the plate to the instructor.

Be gentle in the whole process.

Observe crystals under microscope after 30 minutes or next lab period.


29/61

GEL FILTRATION ON SEPHADEX

INTRODUCTION:

Sephadex is a brand of modified dextrans of microbic origin. Dextrans are high molecular weightpolymers of D-glucopyranose synthesized by a number of bacterial species belonging to the family

Lactobacilleae. These contain almost exclusively !-(1-6)- linkages and are crosslinked withepichlorophydrin to give Sephadex. Various grades of Sephadex are available in which the dextran

contains different degrees of cross linking. The cross links form three-dimensional networks of pores.

The abundance of hydroxyl groups in dextran makes it hydrophilic and causes it to swell in

aqueous solutions to form gels. These gels are able to discriminate against the entrance of moleculesover a certain size into their pores. Columns of Sephadex gels are used to separate and also estimate

the molecular weight of different size protein molecules. Since the pores roughly resemble truncatedcones in shape, the entire volume of the pores is available to small molecules, while larger moleculescan only diffuse part way into the pores. Molecules with an effective hydro-dynamic diameter larger

than the entrance to the pores are, of course, completely excluded from the region within the pores.

Consider a column of Sephadex gel. The total volume, Vt, of the column is the sum of the volumeof water outside the gel, Vo, (called the void volume); the volume inside the pores, V i, (called the

internal volume); and the volume of the gel substance itself, Vg.

Vt= Vo+ Vi+ Vg (1)

In order to travel through the column a molecule must pass through the void volume and a certainfraction, Kdof the internal volume. Therefore, the volume of eluent which must be used to displace a

protein from the top to the bottom of the column is given by:

Ve= Vo+ KdVi (2)

The fraction of the pore volume which is available to any molecule (Kd) depends on the size andshape of the molecule. This fraction is sometimes referred to as the distribution coefficient. For

proteins having similar shapes a single linear function can be used to relate Ve to the log of themolecular weight of the protein. Equation 2 may be used to determine the distribution coefficient,

provided the void and internal volumes are known. The void volume (Vo) can be determined by

measuring the volume of liquid required to elute a substance that is excluded from internal volume ofthe gel. Similarly, the internal volume (Vi) can be determined by subtracting the void volume fromthe volume of liquid required to elute a substance that is small enough to have complete access to the

internal volume.

The internal volume can also be calculated from the known dry weight of the gel (a), and the water

regain (Wr).


30/61

Vi = aWr (3)

In forming the gel the pores in the Sephadex fill with water, and the internal or pore volume is

equal to the volume of water taken up by the Sephadex. The water regain is defined as the number of

grams of water taken up when forming a gel from one gram of dry Sephadex. Sephadex is gradedaccording to its water regain. The grade number divided by 10 is equal to the water regain. Thus, onegram of G-100 has an internal volume of 10 ml. The size of the pores of a Sephadex gel varies

directly with its water regain. Therefore, a greater fraction of the internal volume of Sephadex gelwith a higher water regain will be available to a large protein molecule. See Table I.

When setting up Sephadex columns it is also useful to know its wet density (d, the density of the

gel), and the ml of column bed one obtains per gram of dry Sephadex. The volume occupied by thegel substance itself (Vg) can be calculated from the wet density and the water regain.

Vg 1+Wr

--- = ------- - Wr (4)a d

If the total volume of the column is known, equations 1, 3 and 4 can be used along with the

appropriate data in the table to estimate the void and internal volumes.

A limiting factor in separating a mixture of molecules in a given solution is the volume of thesample

1, 2. The figure illustrates the separation of three materials having different distribution

coefficients. It follows from equation 1, that the volume separating two components (Vs) is equal tothe difference between the distribution coefficients of the components multiplied by the internal

volume. Ideally (broken lines), the concentration of each component in the eluent is equal to itsconcentration in the original sample. Therefore, for complete separation the sample volume, V

x,

should be no larger than Vs. Variations from ideality (solid lines) due to flow irregularities in thecolumn, diffusion and non-equilibrium conditions in the swollen gel, necessitate a reduction in the

sample volume below the value of Vsin order to insure complete separation of the two components.

____________________________________________________________________

1. As long as the viscosity of the sample is not high, the amount of material dissolved in the sampledoes not effect the efficiency of the column. At high viscosities the diffusion of molecules into the gel

network becomes severely impeded.

2. Sephadex contains a small number of carboxyl groups so that the eluent must contain a lowconcentration of ionic species (0.01N) to prevent the protein from being adsorbed by the gel.


31/61

TABLE I - PROPERTIES OF SEPHADEX

ApproximateExclusion Limit

(MW)

Water Regain, Wr(g H2O/g dry gel)

Wet Density(g/cm

3)

Bed Volume(ml/g dry gel)

Sephadex G-25 5,000 2.5 + 0.3 1.13 5

Sephadex G-50 10,000 5.0 + 0.5 1.07 10

Sephadex G-75 50,000 7.5 + 1.0 1.05 12 - 15

Sephadex G-100 100,000 10.0 + 1.0 1.04 15 - 20

Sephadex G-200 200,000 20.0 + 2.0 1.02 30 - 40

ELUTION PROFILE

Effluent Volumes, Ve

SWELLING TIME REQUIRED TO PREPARE SLURRY FROM DRY GEL (HOURS)

25oC 100

oC 25

oC 100

oC

G-25 3 1 G-100 72 3

G-50 3 1 G-200 72 3

G-75 24 2

GEL FILTRATION LAB WORK:


32/61

During two laboratory periods one should accomplish the following:

1. Prepare (pour) a Sephadex G-50 column.

2. Apply a sample mixture to the column for separation.

3. Measure elution volumes of components from the column.4. Make calculations as indicated.

The G-50 (Fine) Sephadex to be used has been equilibrating with water for at least 3 hours whichis adequate swelling time for G-50.

SAMPLE MIXTURE CONTENTS:

1. Blue Dextran (Avg. MW = 1,000,000), a high molecular weight dextran which has covalentlybound to it a blue chromophore.

2.

Cytochrome C (MW = 12,000), an iron-porphyrin protein involved in the respiratory chain.3.

Flavin Mononucleotide - FMN (MW = 514), a coenzyme required in some biological

oxidation-reduction reactions.

Blue dextran is large enough to be excluded from all G-50 pores and FMN is small enoughto pass through all G-50 pores. Cyt C is intermediate in size.

PERIOD I - PACKING THE COLUMN WITH G-50:

NOTE- Pour gel slurry from container as it is provided in lab.Do not transfer to your own beaker.

Do not discard the gel, it will be recovered at the end of the experiment.

Obtain from the instructor a one cm diameter chromatography column with rubber tubing andscrew clamp attached to the bottom, 2 disposable pipets and a rubber pipet bulb. These are to be

returned at the end of the second laboratory period.

Obtain two 1 cm filter papers. Place one at the bottom of the column before pouring the sephadex.Position the disc on top of the porous glass support at the bottom of the column (this is easier to do

with buffer in the column). The paper is needed in order to exclude gel particles from the sinteredglass disc; they tend to pack tightly and severly impede column flow rate.

The other filter disc will be placed on top of the gel after it has settled by allowing it to float down inthe buffer.

Clamp the column on a ring stand, make sure that it is vertical, close the screw clamp and addabout 5 ml of buffer. Add enough G-50 slurry to fill column. A settled column height of about 19 cm


33/61

is desired. Allow the gel to settle with gravity. Use one of the disposable pipets to remove bufferfrom the top of the column when it is necessary to add more gel. Always add additional gel to the

column before the top has completely settled. If it is necessary to add additional gel to the top of thesettled column, layering will generally occur.

NOTE: NEVER allow the buffer level in the column to pass below the surface of the settledgel. If this occurs, air bubbles will be drawn into the column, channeling will result and the columnmust be repoured in order to obtain valid data.

When a settled height of about 19 cm has been attained, commence the buffer flow and the gel will

pack down slightly. Maintain buffer flow until the bed height has stabilized, then collect 10 ml buffer.During this period, with buffer in column above packed bed, carefully place the small circular filter

paper into the buffer and allow it to float down and settle on top of the bed. This protects the gel fromphysical disturbance while applying sample or buffer. Put parafilm on the top of column and store on

benchtop with your name.

PERIOD II - APPLYING SAMPLE TO THE COLUMN:

Allow the eluent level to drop just even with the top of the gel and stop the flow (clamp). Using a

disposable pipet indicated by the instructor add 12 drops of the sample mixture to the top of the gel asfollows. Carefully place the tip of the pipet as close to the top of the bed as possible and still allow a

full drop to leave the pipet. This affords minimum disturbance of the gel when dropping sample. DONOT allow the tip of the pipet to contact the sides of the column either on entry or exit from the

column for contamination will result. BE CAREFUL, the pipet tips are very fragile. Slow and carefulmovement is suggested.

ELUENT COLLECTION:

Eluent collection will begin when the sample goes into the column. Open the clamp and allowsample to flow just into the gel, then stop the flow (clamp). Collect these drops (use a 10 ml

graduated cylinder), they are the first of the measured total volume. Wash sides of column close tosurface of the gel where sample has been touching the glass with a small amount of buffer (certainly

no more than the volume of the applied sample). The object of this wash is to get the thin film ofsample still on the glass into the gel in a minimum volume. Allow the wash to flow into the column in

the same manner as the sample. Repeat washing procedure once more then fill column with bufferand continue elution. Attempt to keep the column full of buffer while eluting to afford a static head

pressure.

Record total volume collected from the start to the first drop of each component eluted. Eachreadings represent the eluting volume, Ve, for the component eluted. When finished, elute out last

component completely , then return the gel to beaker from which it was obtained.

MEASUREMENTS AND CALCULATIONS:


34/61

1. Measure the total volume of eluted buffer to the point where the first component begins to flow

from the column. This volume is the experimental Vo.

2. Continue to collect and measure volume. Record the total volume (from the beginning) at

which Cyt C first appears in the eluent. This volume is Ve(eluting volume) and from it Kdfor CytC can be calculated.

3. Continue measuring total buffer from column. Record the volume collected from the start tothe point where FMN first elutes from column. This volume represents the total of Voand Viand

thus affords an experimental value for Vi.

4. Calculate Vt from physical measurement of the packed gel (the volume of a cylinder).

5. Calculate the dry weight (a) of the gel. (See table for conversion factor).

6. Calculate Vi using equation 3 from the dry weight (a) and the water regain. Compare with theexperimentally determined Vi.

7. Calculate Vg using equation 4.

8. Calculate Vo using equation 1 and compare with the experimentally determined Vo.

9. Calculate Kdfor Cyt C using equation 2. Do this twice, first from items 1, 2 and 3 above and

second from items 2, 6 and 8 above.


35/61

WORKSHEETGEL FILTRATION Name ___________________

Other members of your group: __________________________________

Inside diameter of column: _____________. Height of gel after settling: _____________.

Volumes recorded during run: (With sample setting on top of the column the volume = 0 ml)

Total from column start to 1st drop of the 1st component: ____ ml. Color = _________

Total from column start to 1st drop of the 2nd component: ____ ml. Color = _________

Total from column start to 1st drop of the 3rd component: ____ ml. Color = _________

Vo(experimental) ______ml; Ve(2nd component) ______ml; Vi(experimental) _______ml

Vt(column packing) __________

a (dry weight) ____________

Vi(equation 3) ____________

Vg(equation 4) ____________

Vo(equation 1) ____________

Kd(equation 2) ____________


36/61

QUESTIONS: (include the questions and your answers in your notebook at he end)

1. In your own words, explain how molecules are separated using gel chromatography.

2. Why is it necessary to use a buffer eluent instead of pure water during gel chromatography ?

3. To what class of biomolecules does Blue Dextran belong ? _________________________

4. To what class of biomolecules does Cytochrome C belong ?_________________________

5. What is the nonprotein group associated with cytochrome C ? __________________

6. Draw the structure of FMN.


37/61

INVERTASE (!-FRUCTOFURANOSIDASE)E.C. 3.2.1.26

Reference: Melius, P., J. Chem. Ed., 48(11) 765-66 (1971)

Invertase is an enzyme which catalyzes the hydrolysis of the glycosidic bond of sucrose to giveglucose and fructose. The following laboratory periods will be devoted to isolating, purifying and

studying the properties of invertase.

InvertaseSucrose ------------> Glucose + Fructose

PERIOD I - Growth, isolation and purification.

NOTE: 24 hours prior to the start of your scheduled laboratory period a representative from

each group needs to commence the yeast incubation.

YEAST PREPARATION: (24 hours prior to scheduled lab period)

Weigh out 2.8 gm Fleischmanns dry yeast and transfer to a 50 ml Erlenmeyer flask. Add 10 ml 0.1

M NaHCO3buffer using a graduated cylinder and mix with a stirring rod until smooth (not lumpy).Stopper with cotton and incubate in environmental chamber at 37

ofor 24 hours.

WORK-UP YEAST PREPARATION: (Perform during regular scheduled lab period)

Part of the group should proceed with centrifugation of the yeast mixture while at the same timeothers in the group prepare the chromatography column.

CENTRIFUGATION:

Obtain two 15 ml centrifuge tubes from the instructor. After the 24 hour incubation period hasended, transfer the incubation mixture into one of the centrifuge tubes and balance the tubes by adding

water to the other tube. Use the two pan balance provided in the lab to ensure that the tubes arebalanced against each other. To do this adjust the balance to be used to the zero mark before placing

the tubes on the balance. ---THIS IS IMPORTANT. UNEQUAL WEIGHT DISTRIBUTION INTHE CENTRIFUGE HEAD MAY DAMAGE THE CENTRIFUGE OR YOU.--- Place the two

balanced tubes opposite each other in the centrifuge head and note the numbers of the holes into whichthe tubes were placed. Centrifuge for 15 minutes at 10,000 x g at 4

oC. After centrifuging, carefully

remove the tubes from the centrifuge head to avoid mixing the compacted solids (pellet) with thesolution (supernatant). Decant the supernatant into a clean 10 cc graduated cylinder or transfer using


38/61

a disposable pipet such that none of the solid material passes with the supernatant. Label this yeastextract "crude invertase" and record the volume. Discard the centri-fuge tubes.

CHROMATOGRAPHY:

A sample of crude invertase will be chromatographed on a column of Sephadex G-200 to separatethe invertase from the other protein material present. Prepare a column of G-200 using the procedure

learned in the gel filtration experiment. Use 0.05 M acetate buffer pH 4.7 in packing and eluting thecolumn. The packed bed should be approximately 18-19 cm in height. When the packed height has

stabilized, place 1 ml of crude invertase on the column, again using the techniques of sampleapplication practiced in the GEL FILTRATION experiment.

After washing the sample into the column, add buffer, elute the column and collect the material

eluting from the column as follows: Collect 10 fractions of approximately 1-1.5 ml each (this shouldrepresent almost everything up to a pale yellow band which will be migrating down the column.)

Then collect 4 fractions of 3-4 ml each. Elution should require 1-1/2 hour and the bulk of the proteinin the yeast extract will trail the enzyme (invertase) down the column as a pale yellow band.

Keep and refrigerate all fractions until the activity determination has been completed (Period III). See

instructor for storage. Also, keep the remainder of your crude invertase (not chromatographed) withthe other tubes.


39/61

PERIOD II. PREPARATION OF STANDARD PLOT:

(Absorbance vs. Sugar Concentration) ALL TEST TUBES USED IN SUBSEQUENT STEPSMUST BE CLEAN AND DRY. This colorimetric assay is based on the color produced when glucose

is treated with dinitrosalicylate.

Solutions of known glucose concentration will be treated at timed intervals with 2.0 ml of 3,5-dinitrosalicylate (DNS) reagent, followed by heating for exactly 5 minutes in a boiling water bath. Remove

and immediately cool the tubes to room temperature using tap water. Then add 15 ml of distilledwater to each tube using a buret, mix well and measure absorbance at 540 nm (A540) using a Milton

Roy 501 spectrophotometer. Record absorbances for each of the samples and for the blank. Thesugar standard is a solution of 0.005 M Glucose plus 0.005 M Fructose.

PREPARATION OF SAMPLES FOR STANDARD(volumes are in ml, add reagents in the orderlisted):

Sample tube # 1 2 3 4 5 6 7

Sugar Standard (G+F) 0 0.2 0.4 0.6 0.8 1.0 1.2

0.3 M Sucrose 1.0 1.0 1.0 1.0 1.0 1.0 1.0water, distilled 2.0 1.8 1.6 1.4 1.2 1.0 0.8

Add DNS then:

Place into l00obath

at Timer reading (min): 0 2 4 6 8 10 12

Remove from and coolat Timer reading (min): 5 7 9 11 13 15 17

PIPETS: 50 ml buret.................waterpipettman...........glucose + fructose

pipettman.................sucroseRepipet......................DNS

USE OF SPECTROMETER:

Turn on instrument and allow to warm up.Set wave length to 540 nanometers (nm)

Set to Absorbance (A) and press the auto zero on blank.Without making further adjustments determine absorbance of samples proceeding in order

from the most dilute to the most concentrated sample.Obtain disposable cuvets (2), one for the blank and the other for the samples.

Carefully align the cuvets the same way in the holder for each reading.


40/61

DATA & PLOT:

Record all data in your notebook. Calculate and tabulate values for net absorbance (A540of asample - A540of the blank). This represents the amount of glucose/fructose actually producing the

absorbance. The solution containing equal amounts of 0.005 M glucose and 0.005 M fructose is

equivalent to a solution of 0.005 M sucrose before hydroysis. Therefore, the A540values from abovecan be equated to amounts of sucrose hydrolyzed. e.g. 0.4 ml of the glucose/fructose stock solutionwould be equivalent to 0.005 M x 0.4 ml = 0.002 millimol of sucrose hydrolyzed. Make a standard

plot of net A540vs micro mol of sucrose hydrolyzed. The line drawn must go through the coordinates0,0 because the net absorbance in absence of any standard must be zero. The plot should be taped into

your notebook and included with all other data.


41/61

INVERTASE WORKSHEET (example data for notebook) Be sure to use subheadings and

describe everything adequately. Remember, someone else should be able to reproduce your

work with nothing other than your lab notebook!

Other group members:

1. Data from incubation and centrifugation.

2. Data from chromatography.

3. Write structural formulas showing the reaction which invertase catalyzes.

4. Calculate Vofor the column.


42/61

INVERTASE (!-FRUCTOFURANOSIDASE)

E.C. 3.2.1.26

PERIOD III & IV: Column Profile by Enzymatic Activity Assay and Total Protein

The "activity" of an enzyme refers to the catalytic effect exhibited by the enzyme. An enzymeassay refers to the reaction or means utilized to detect or measure the activity of an enzyme. The

activity of invertase refers its catalysis of the hydrolysis of sucrose to yield glucose and fructose.Invertase activity is assayed by using dinitrosalicylate (DNS) which reacts quantitatively with the

reducing sugar produced during hydrolysis. The amount of reaction product (and therefore thereducing sugar) is determined by using a Spectrometer and a standard plot from the previous lab.

A sample from each of the 14 fractions will be assayed in Part 1 and 2. The purpose of these

assays is to determine which of the chromatographic fractions collected contain the most invertaseand, also, to estimate the total protein in each fraction. The "active" fractions will be combined and

used for the Kmand Vmdetermination in Period V.

PART I: DETERMINATION OF ENZYME ACTIVITY IN COLUMN FRACTIONS

(Enter data in your notebook.)

Obtain the column fractions which have been stored in the refrigerator (Do not discard any ofthese, you will need some of them later.)

Set up and label a series of 14 test tubes (one corresponding to each fraction from the column). Have

these tubes incubating at room temperature during preparation. Also, have the sucrose, which will beadded to the tubes later, equilibrating at room temperature. This is done so that when the sucrose is

added the reaction begins immediately at room temperature and there is no temperature fluctuation.

To each of the 14 tubes, add 0.7 ml of 0.05 M acetate buffer, 1.2 ml of water and 0.1 ml of thecorresponding column fraction. The assay of the first column fraction will represent the blank used to

correct the Abs540 of the other 13 assays.

Use a 10 ml buret to measure the acetate buffer, a 50 ml buret for measuring water and disposablepipets for transferring the column fraction samples. Two full drops from a disposable pipet is

approximately 0.1 ml, so add that number of drops to each reaction tube. Use a clean disposable pipetfor each of the enzyme fractions (14 pipets will be needed) or use a micropipet (eppendorf, etc.).


43/61

Sucrosewill be added to initiate the reaction. Organization and timing are critical, therefore, plan

carefully before proceeding.

To proceed with the assay, add 1.0 ml of 0.3 M sucrose (already at room temp) to each tube and mix.

Each tube is incubated at room temp. for exactly 5 min. (timers are provided by the instructor). Theenzyme is acting on sucrose during this incubation.

The enzyme reaction is stopped by pipetting 2 ml of 3,5-dinitrosalicylate (DNS) reagent into each tubeat the end of its 5 minute incubation period and mixing (NOTE: Removal of tubes from the bath will

not terminate reaction. The 5 minute incubation must be terminated by addition of DNS reagent).

Transfer the tubes to a boiling water bath for 5 minutes to develop the DNS color. Then immediatelycool to room temperature in water or color development will be inconsistent.

Dilute by adding 15 ml of water to each tube, mix well and measure the A540as with standards.

Determine micromoles of sucrose hydrolyzed by using these absorbance readings and the slope from

the standard plot prepared in period II, then prepare a graph of units of enzyme activity (1 unit = 10-6

moles sucrose hydrolyzed/minute) vs. fraction number eluted from the column. This yields an activity

profile of the column eluate.

Combine those 2-3 fractions of invertase which represent the major part of the activity as indicatedfrom the assays, label this tube "purified invertase" and identify by group number. This "purified

invertase" will be used to prepare a stock solution for the Kmand Vmdetermination.

PART II: DETERMINATION OF TOTAL PROTEIN IN COLUMN FRACTIONS

Proteins other than invertase in your extract were also eluted during chromatography. Sinceessentially all proteins (including invertase) contain tryptophan and tyrosine, essentially all

proteins absorb UV radiation at 280nm. You can assess the total protein by measuring theabsorbance at 280nm of each of your column fractions. Measure the Abs of each sample at this

wavelength and construct a double y plot in excel where one y-axis represents total protein (Absat 280nm) and the other y-axis represents the amount of enzyme activity. The x-axis will

represent your column fractions. Keep in mind, you may need to do a 1:10 dilution of yourfraction when measuring the Abs(280nm) since it may be outside the linear range of the

instrument. If you do a 1:10 dilution, dont forget to multiply the Abs by 10 when constructingyour plot..


44/61

INVERTASE WORKSHEET (This data should be in your notebook in this tabular format)

1. Data from determination of enzyme activity (enzyme assays).

Fraction # 1 2 3 4 5 6 7 8 9 10 11 12 13 1

Buffer, ml

Water, ml

Enzyme, drops

Time, start

Time, add DNS

Time, start 100o

Time, stop

Dilute, ml

Abs 540

Net Abs 540 0

mol sucrose

mol/min

2. Slope of the standard plot from Period II. ______________

3. Fraction numbers pooled and retained. __________


45/61


46/61

Example Double Y Plot


47/61


E.C. 3.2.1.26

PERIOD V:Determination of Kmand Vmax.

DETERMINATION OF KmAND VmaxFOR INVERTASE:

A portion of the combined enzyme fractions must be diluted to be in the proper working range.

Dilute 1 ml of your pooled enzyme to xx ml with acetate buffer (graduated cylinder is goodenough here). This diluted invertase is then the enzyme stock solution for the kinetic assays.

BURET AND PIPET USE:

50 ml buret......................water

1 ml micropipet...............acetate buffer1 ml micropipet...............sucrose

1 ml micropipet...............enzyme1 ml micropipette or pipetteman ............................dinitrosalicylate (DNS)

PREPARATION OF SUBSTRATE STOCK SOLUTIONS:

Prepare a series of dilutions of sucrose from the 0.3 M sucrose stock solution available in thelab as shown in the table below. Each must be well mixed. See Table I.

TABLE I

Sample solution # 1 2 3 4

0.3 M Sucrose, mL 2 1 1 1

Distilled H2O, mL 4 4 6 8

Final conc., M 0.1 0.06 0.043 0.033

These sucrose solutions represent different substrate concentrations and a reaction velocity will bedetermined at each substrate concentration as indicated in the following section.

PREPARATION OF REACTION TUBES:

Prepare these tubes adding reagents in the order listed in the table (NOTE: The blank contains

no enzyme). Incubate each at room temperature for EXACTLY 5 minutes. Precision in timingand adding reagents should be analogous to previous assays. Terminate the reaction by adding 2


48/61

ml of DNS reagent to each tube, mix well and heat in boiling water bath for 5 minutes. Cool toroom temperature, add 15 ml of water to each tube and measure the A540as before. See Table II.

TABLE II

Assay tube # 1 2 3 4 5 6

0.05 M acetate, ml 1 1 1 1 1 2

Enzyme, ml 1 1 1 1 1 0

Sucrose,Add 1 ml of: 0.033 M 0.043 M 0.06 M 0.10 M 0.3 M 0.3 M

PLOTS & CALCULATIONS:

Determine the micromoles of sucrose hydrolyzed/min from the absorbance using the standardplot. Prepare a Lineweaver-Burke plot using the data from each reaction tube where the units of

velocity, v, are micromoles sucrose transformed/min and the units of substrate concentration, S,are M. From this plot and the relationships of the Lineweaver-Burke equation determine the

values for Kmand Vm.


49/61

Kmand VmaxINVERTASE DATA(recorded in your notebook in your data section)

1. Lab data from the enzyme rate vs. substrate study. Prepare tabular with 6 columns. Rows

prepared as follows:

a. Tube No. (6 tubes thus, 6 columns in the table)b. Time, start

c. Time, DNS addedd. Time, stop

e. Abs (540nm)f. Net Abs (if applicable, but not applicable on a dual beam instrument)

g. mol sucrose (abs/slope)

h. v (moles sucrose/5 min.)i. 1/v

j. [S]

k.

1/[S]l. M

2. Values for Kmand Vm.

3. The concentration of your invertase from Bradford and Plate Reader experiment __________

4. Maximum activity of invertase ("most active fraction"):

a. Maximum activity (Vmax) from Item 2 above = ________ mol/min

5. Specific activity of invertase ("most active fraction") (please remember the dilution factor):

mol substrate transformed/minSpecific activity = ----------------------------------------

mg enzyme

Specific activity = _________

6. Draw the structure for 3,5-dinitrosalicylic acid. You should know this for your quiz, as well as

all other pertinent structures.


50/61


E.C. 3.2.1.26

PERIOD VI: Inhibition of invertase with p-nitrophenol. Determination of Kmand Vmand Ki

DETERMINATION OF KmAND VmFOR INVERTASE:

A portion of the combined enzyme fractions must be diluted to be in the proper working range. Your

Invertase solution from the last experiment, Det. of Kmand Vmshould be sufficient as solution ESI inthis experiment. If not, prepare solution ESI by diluting your most concentrated invertase fraction to

10 ml with water (graduated cylinder is good enough here) such that the final absorbance will begreater than or less than that used in your last experiment, as appropriate. ESI is the enzyme stock

solution to be used for the kinetic assays Tables II, III & IV). Alternatively, if there is no morepurified invertase available, make10 milliliters of a 1 mg/mL solution in a 10 mL vol. flask using solid

invertase. Measure the absorbance of this solution at 280 nm. Dilute to an A280of 0.1 this is yourworking enzyme stock solution. If the absorbance is not sufficient, add a few more crystals to your

volumetric flask, dissolve and measure again.

BURET AND PIPET USE:

50 ml buret..........................water1 ml Eppendorf pipet...........acetate buffer, sucrose stock solutions, sucrose dilutions,

enzyme, PNPI and PNPII10 mL serological pipet..PNP (inhibitor) stock solutions

Repipet................................dinitrosalicylate (DNS)

PREPARATION OF SUCROSE (SUBSTRATE) STOCK SOLUTIONS:

Prepare a series of dilutions of sucrose from the 0.3 M sucrose stock solution available in the

lab as shown in the table below. Each must be well mixed. See Table I.

TABLE I

Sample solution # 1 2 3 4

0.3 M Sucrose, ml 3 1 1 1

Diltilled HOH, ml 6 4 6 8

Final conc., M 0.1 0.06 0.043 0.033

These sucrose solutions represent different substrate concentrations and a reaction velocity will be


51/61

determined at each substrate concentration as indicated in the following section.

PREPARATION OF REACTION TUBES FOR ASSAYS:

Prepare these tubes adding reagents in the order listed in the table (NOTE: The blank contains waterinstead of enzyme). Incubate each at RT for exactly 5 minutes. Precision in timing and addingreagents should be analagous to previous assays. Terminate the reaction by adding 2 ml of DNS

reagent to each tube, mix well and heat in boiling water bath for exactly 5 minutes. Cool to roomtemperature, add 15 ml of water to each tube and measure the A540as before. See Tables II, III & IV.

PREPARATION OF REACTION TUBES FOR UNINHIBITED ASSAYS:Determine Kmand Vm.

TABLE II

Assay tube # 1 2 3 4 Blank

0.05 M acetate, ml 1 1 1 1 1

Sucrose, Add 1 ml of: 0.033 M 0.043 M 0.06 M 0.10 M 0.10 M

Enzyme, ml 1 1 1 1 1 ml HOH

PREPARATION OF TWO INHIBITOR SOLUTIONS:

Pour 20 mL 0.08 M PNP from the stock provided into a clean 30 mL beaker. Prepare twoconcentrations of p-nitrophenol from the 0.08 M PNP stock solution. Measure these volumes

accurately with a serological pipet and a 10 mL volumetric flask. Make PNPI by diluting 9.0 ml ofPNP stock to 10 ml using 0.05 M acetate buffer and similarly make PNPII by diluting 7.0 ml of PNP

stock to 10 ml using 0.05 M acetate buffer. PNPI and PNPII are the inhibitor solutions to be used inthe assays below.

PREPARATION OF REACTION TUBES FOR ASSAYS AT [I] = 0.072 M:TABLE III


PNPI, ml 1 1 1 1 1



PREPARATION OF REACTION TUBES FOR ASSAYS AT [I] = 0.056 M:


52/61

TABLE IV


PNPII, ml 1 1 1 1 1



PLOTS & CALCULATIONS:

Determine the micromoles of sucrose hydrolyzed/min from the absorbance using the standard plot.

Then prepare a Lineweaver-Burke plot using the data from each reaction tube where the units ofvelocity, v, are micromoles sucrose transformed/min and the units of substrate concentration, S, are

M. From this plot and the relationships of the Lineweaver-Burke equation determine the values for Kmand Vm.


53/61

INVERTASE DATA (Typical data which should be in your notebook - titled)

1. Absorbance data for ESI (if applicable to this lab or state the enzyme solution used).

2. Show the dilution calculation if applicable.

3. Include: v, [S], 1/v, 1/[S] and Abs(540 nm) data (in tablular form) resulting from the

assays in Table II, Table III and Table IV. Plot all data on one L-B Plot.

4. Values for Kmand Vmfrom the Lineweaver-Burke plot.

5. Based of the L-B plot, what type inhibition is exhibited by p-nitrophenol ? ___________

6. Calculate Ki from each [I] and its respective Km(app).


54/61


55/61

#

Visualizing the protein bands:

Once the gel is run, remove the gel cassette from the apparatus and remove the gel from the

cassette using an opener. Transfer the gel into a suitable container

Wash SDS from gel with three consecutive washes (10 min each) in distilled water. Add enough

Colloidal Coomassie stain (20% ethanol, 1.6% phosphoric acid, 8% ammonium sulfate, 0.08%

Coomassie Brilliant Blue G-250) to cover gel.Incubate at room temperature on rotary

agitator for at least twelve hours.

Decant stain and rinse gel twice with distilled water (1 min each).

Destain gel with distilled water until background is low (4 hours).


56/61

$

Solution Preparation (SDS PAGE)

1X Running buffer (5 L): 0.025 M Tris, 0.192 M Glycine, 0.1% SDS

Tris base 15.0 g

Glycine 72.0 g

SDS 10.0 g

Dissolve in 500 mL of distilled water in a beaker with stirring. Do not adjust the pH. Mix with

4.5 L of distilled water to get 5 L of 1X running buffer. Final pH will be ~8.3.

Destaining solution (2 L): 10% Acetic, 45% Methanol

Distilled water 900 mL

Methanol 900 mL

Acetic acid (glacial) 200 mL


57/61

Chem4650/5650 General Biochemistry Lab

Qualitative and Quantitative Analysis of Invertase

Lab-on-a-Chip Technology

Although protein analysis technologies are developing fast, the current standard

method for protein sizing is still denaturing sodium dodecylsulfate-polyacrylamide gelelectrophoresis (SDS-PAGE). As you will recall from last semester, SDS-PAGE is an

electrophoretic separation of proteins in a polymeric material. The proteins are coated in

SDS which gives them an overall negative charge, and they are then loaded into a well in

an SDS-PAGE gel where they migrate towards the positive electrode. Separation is based

on molecular weight.

Many researchers rely on this traditional, labor-intensive, and time-consuming

electrophoretic method that has not substantially changed in the past 30 years. With the

increasing focus on proteins, there is a strong demand to automate and speed up protein

analysis. A significant investment was made to develop more automated methods (e.g.,

capillary gel electrophoresis), which, however, never replaced SDS-PAGE. The recent

development of microfluidic or lab-on-a-chip analysis systems offers an alternative for

protein analysis and has therefore stimulated a lot of academic and industrial research.

Microfluidic technology allows for the active control of fluids in microfabricated

channels that are only a few micrometers in dimension and which have no moving parts.

These chips can contain the emulation of pumps, valves, and dispensers for sample

handling on the chip, a separation column for electrophoretic separation, and a reaction

system. Microfluidic technology aims to integrate several sequential experimental steps

into one process to obtain a complete laboratory on a chip. Recently, the first commercial

lab-on-a-chip analysis system, the Agilent 2100 bioanalyzer (Agilent Technologies

Deutschland GmbH), which was developed in collaboration with Caliper Technologies

Corp. (Mountain View, CA), was introduced, and is the subject of this exercise. This

system allows for the rapid, automated electrophoretic separation of proteins and the

integration of multiple experimental procedures, such as sample handling, separation,

staining, destaining, detection, and analysis into a single process. This white paper

demonstrates the performance of the chip-based protein analysis in terms of resolution,

sensitivity, linear dynamic range, sizing, and quantitation in comparison to conventional

SDS-PAGE and protein quantitation methods.

Source: Journal of Biomolecular Techniques 13:172178 2002 ABRF


58/61


59/61

Kit: Protein 200 Plus kitAssay: Protein 200 Plus assayApplication:A 18 kDa protein was purified using affinitiy chromatography. The starting materialand the column fraction were analyzed with the protein assay. The protein of interest wasdetermined to be 99% pure and the concentration in the column fraction was 167 ng/l.The protein assay allows protein purity and concentration to be determined in one step, inaddition it calculates protein size for reconfirmation.Courtesy of P. Sebastian and S.R. Schmidt GPC-Biotech AG, Martinsried, GermanyCorresponding application note: data not published

The Protein 200 Plus LabChip kit is a fast and reliable assay capable

of quantifying and sizing a multitude of different protein samples.Used with the Agilent 2100 bioanalyzer it can analyze ten, 4 l samplesin less than 30 minutes.

Protein Quantitative Comparison ApplicationAbsolute protein quantitation


60/61

Kit: Protein 200 Plus kit

Assay: Protein 200 Plus assayApplication:A comparative analysis of different techniques used for absolute proteinquantitation was performed analyzing 3 different proteins (CA, BSA, OV) in 4 differentconcentrations (40 - 1250 ug/ml). The same samples were quantitated using the

Agilent 2100 bioanalyzer, two commonly used total protein quantitation assays, Lowry andBradford, and SDS-PAGE, stained with Coomassie. The relative standard deviation (CV)and the error compared to the target concentration were determined. A comparison shows thatthe CV and error for the Agilent 2100 bioanalyzer are better than for the SDS-PAGE by a factorof 2. This data demonstrates that the Agilent 2100 bioanalyzer is a viable alternative for proteinquantitation. It allows the quantitation of individual proteins and simultanous determinationof protein purity and size.


61/61

Comparison to SDS-PAGE

The performance of the Agilent 2100 bioanalyzer, the first commercial lab-on-a-chip

system, and the Protein 200 Plus LabChip kit is compared with conventional protein

analysis techniques such as SDS-PAGE, Lowry, or Bradford. Lab-on-a-chip technologyfor protein analysis allows for the integration of electrophoretic separation, staining,

destaining, and fluorescence detection into a single process, and for it to be combinedwith data analysis. The chip-based protein assay allows purity analysis, sizing, andrelative quantitation based on internal standards or absolute quantitation based on user-

defined standards.The chip-based protein analysis is comparable in sensitivity, sizing

accuracy, and reproducibility to SDS-PAGE stained with standard Coomassie. Resolution

and linear dynamic range are improved. Absolute quantitation accuracy andreproducibility is improved in comparison to SDS PAGE and is comparable to batch-

based quantitation methods such as Lowry and Bradford.The lab on-a-chip system has

several additional advantages over conventional SDS-PAGE including fast analysis

times, reduced manual labor, automated data analysis, and good reproducibility. Withsuch a system, the protein of interest can be tracked during the whole purification

procedure, for example, from cell lysates through column fractions to purified proteins.

Source: Journal of Biomolecular Techniques 13:172178 2002 ABRF

FIGURE 1Resolution of the chip-based analysis. The size resolution for the separation of a protein mixture of eightdifferent proteins was compared showing the gel-like image and the electropherogram from the chip-basedanalysis and the gel image and the gel scan from a 420% gradient gel The molecular weights are shown

BioChem Procedures S15

Documents

Transcript of BioChem Procedures S15