Analysis of Carbohydrates and Lipids

from Eggs
CHEM 4581: Biochemistry Laboratory I Version: March 25, 2008

BACKGROUND AND GOAL

Carbohydrates. In the field of biochemistry, carbohydrates represent one major class of biological
macromolecules. There are five classifications of carbohydrates: monosaccharides, disaccharides,
oligosaccharides, polysaccharides and nucleotides. Monosaccharides can be composed of a 5-
membered ring, as in fructose, or a 6-membered ring, as in glucose or sucrose.

Classification Example Distinguishing Feature

Monosaccharides Fructose 5-membered ring
Glucose 6-membered ring

Dissaccharides Sucrose

Oligosaccharides Raffinose 3-10 monosaccharides

connected by a glycosidic bond

Polysaccharides Amylose Thousands of monosaccharides

Glycogen connected linearly and in branched
form

However, carbohydrates (or sugars) are commonly found covalently linked to proteins. When sugars
are attached to proteins, the proteins are called “glycoproteins”. In most cases, crystal structures of
proteins will not contain carbohydrates that may be present in in vivo systems. This is because the
carbohydrates often cause disorder in protein structure, prohibiting crystallization. So, proteins that
have been isolated from natural sources would be deglycosylated prior to crystallization attempts.
As a result, it is important to know whether a protein is a glycoprotein or not.

In Part 1 of this study, students will determine whether or not two highly studied proteins –
lysozyme and ovalbumin - are glycosylated using the phenol-sulfuric acid assay with any two of the
following sugars as assay standards – sucrose, glucose, galactose, or dextrose.

Lipids. Lipids (or fats) can be classified into five major groups – fatty acids, triacylglycerols,
glycerophsopholipids, sphingolipids, and cholesterol and its derivatives. Lipids are well known to be
a major component of egg yolks. In Part 2 of this study, students will study two types of lipids –fatty
acids and glycerophospolipids – from hen or quail egg yolks. Students will convert fatty acids into
fatty acid methyl esters and analyze these derivatives via GC-MS. Phospholipid analysis will be done
via thin-layer chromatography (TLC). Students will determine the composition of fats in each type
of egg yolk and how they compare.

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 MATERIALS AND REAGENTS

Carbohydrate Study

Hen Egg White Lysozyme, Sigma

Ovalbumin, Sigma

Phenol

Sulfuric Acid

Galactose

Sucrose

Dextrose

Glucose

UV-1601 Spectrophotometer, Shimadzu

Quartz Cuvettes, or disposable cuvettes

Lipid Study

Chicken eggs

Quail eggs

Acetone

Chloroform

Methanol

Phospholipid Standards
o Phosphatidylcholine
o Phosphatidylethanolamine
o Phosphatidylinositol, if available

250-mL Round-bottom Flask

Silica gel TLC plates (with plastic backing)

Iodine chips

Sulfuric Acid

Nitrogen gas Cylinder

Ultra High Purity (UHP) Helium cylinder (for GC-MS)

70°C water bath

Hexane

GC-MS (from Organic lab, record manufacturer and model in lab notebook)

Fatty Acid Methyl Ester Standards
o Linoleic Acid Methyl Ester
o Myristic Acid Methyl Ester
o Oleic Acid Methyl Ester
o Palmitic Acid Methyl Ester
o Stearic Acid Methyl Ester
o Tridecanoic Acid Methyl Ester

Hamilton Syringe

LABORATORY ADMINISTRATION

Students will work in pairs or individually. Staggered start times will be necessary to avoid a
bottleneck for use of the GC-MS. TA’s will give specific directions on when students should report to
lab. (Usually 45-min intervals are sufficient.) Students are expected to know how to use the GC-MS
in the Organic Chemistry Laboratory. Please see your TA if there are any concerns.

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 EXPERIMENTAL PROCEDURES

Carbohydrate Content in Egg White Proteins

1. Prepare 10 mL of 10 mg/mL ovalbumin in a 15 mL conical tube. Dissolve the ovalbumin in dI

water.

2. Prepare 20 mL of 5% (w/v) phenol in a glass container by dissolving the appropriate amount of

phenol in water. Perform this step in the fume hood. CAUTION: Phenol is highly toxic and
flammable. Wear latex gloves and work in the fume hood when handling phenol-containing
solutions. Dispose of phenol liquid waste in designated containers.

3. Wash 15 large test tubes. Try your best to dry each tube, but do not worry if you are not able to
fully dry them. Label each tube according to the sample name in Table 1. You will perform two
standard curves using galactose, sucrose, glucose or dextrose.

4. Prepare your reaction mixtures according to Table 1. Add the phenol to each sample then mix
with gentle swirling in the fume hood. Then add the sulfuric acid using a serological pipette and
mix again with gentle swirling. CAUTION: This reaction is highly exothermic!

5. Allow the reaction mixtures to sit at room temperature for 20 minutes in the test tubes in the
fume hood. Afterward, transfer some of the reaction-mix into labeled cuvettes for the remaining
5 minutes of the incubation period. You may transfer by pouring or by pipetting using glass
Pasteur pipettes. Do not expose the reaction mix to any plastic or the rubber bulbs!

6. Measure the absorbance spectrum from 450-550 nm to identify the peak wavelength. Record the
peak wavelength to the nearest whole number and absorbance for the standards at that
wavelength.

7. Record the absorbance of the standards and protein samples at the peak wavelength.

8. Plot your absorbance data vs. amount of carbohydrate in
g and put your data in your lab
notebook. Determine the amount of carbohydrate in each protein sample using each standard
curve. If the absorbance value is outside of the standard curve range, then report the amount as
being <20
g or >100
g.

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 Table 1. Preparation and Absorbance Measurements for Glycoprotein Content in
Lysozyme and Ovalbumin

Volume of
Volume Volume of Volume of
Sample Sulfuric Abs
of Stock* dI Water 5% Phenol
Acid
Reference None 1 mL 1 mL 5 mL

Blank None 1 mL 1 mL 5 mL

20
g Sugar #1 0.2 mL 0.8 mL 1 mL 5 mL

40
g Sugar #1 0.4 mL 0.6 mL 1 mL 5 mL

60
g Sugar #1 0.6 mL 0.4 mL 1 mL 5 mL

80
g Sugar #1 0.8 mL 0.2 mL 1 mL 5 mL

100
g Sugar #1 1 mL None 1 mL 5 mL

20
g Sugar #2 0.2 mL 0.8 mL 1 mL 5 mL

40
g Sugar #2 0.4 mL 0.6 mL 1 mL 5 mL

60
g Sugar #2 0.6 mL 0.4 mL 1 mL 5 mL

80
g Sugar #2 0.8 mL 0.2 mL 1 mL 5 mL

100
g Sugar #2 1 mL None 1 mL 5 mL

10 mg/mL
0.2 mL 0.8 mL 1 mL 5 mL
Lysozyme
10 mg/mL
0.2 mL 0.8 mL 1 mL 5 mL
Ovalbumin
*Prepare carbohydrate standard stock solutions to be 0.1 mg/mL.

Peak Wavelength = nm

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Isolation of Phospholipids in Egg Yolks

1. You will be provided with either one chicken egg or two quail eggs. Carefully separate the yolk
from the white. Discard the white, and place half of a chicken yolk or two quail yolks into a 15-
mL centrifuge tube. If using chicken egg yolk, then give the other half to another student.

2. Fill the centrifuge tube with acetone (which is very flammable) and stir thoroughly. You should
obtain a large quantity of solid precipitate.

3. Centrifuge the slurry in the clinical centrifuge on setting "7" for 1.5 minutes. Retain the pellet.
Discard the supernatant.

4. Add ~10 mL of acetone to the tube and mix thoroughly (using a small spatula since the pellet is
very thick and viscous). Centrifuge for 1 minute on setting "7". Discard the supernatant.

5. Repeat step 4 two more times.

6. Add 10 mL chloroform/methanol (2:1 v/v) to each tube, mix thoroughly and centrifuge for 1
minute on setting "7". Transfer the supernatant to a 250-mL round bottom flask.

7. Repeat step 6 two more times. Pool the supernatants in the round bottom flask.

8. Cover the opening of the round bottom flask with foil. Poke a hole into the top of the foil that will
fit the Pasteur pipette connected to the N2 gas.

9. Concentrate the extract to near dryness under a N2 stream of gas in the fume hood. Set the N2
flow to the lowest pressure possible. When drying, the supernatant becomes very cold, so the
flask should be heated in a water bath during the drying step. Stop the evaporation when the
solution becomes viscous or ~1 mL of sample is left.

10. Using a pasture pipette, transfer the concentrated solution to a 15 mL centrifuge tube. If the
sample is too viscous or has dried out, it may be necessary to add a small amount of chloroform
to remove it.

11. Add 10 mL of acetone to the centrifuge tube to precipitate the phospholipids. Vortex the sample
thoroughly, then centrifuge for 1 minute on setting "7". Discard the supernatant, save the pellet,
which contains the precipitated phospholipids.

12. Dissolve about 15 mg (about the size of a small glob on the tip of the spatula) of precipitated
phospholipids in 2 mL of chloroform. You will use a portion of this phospholipid solution for
phospholipid analysis by TLC and for hydrolysis.

Hydrolysis of Fatty Acids and Conversion to Fatty Acid Methyl Esters (FAMEs)

13. Place 0.2 mL of phospholipid solution in a 15 mL centrifuge tube.

14. Add 2 ml of 5% H2SO4 in CH3OH. (CAUTION: Strong Acid! Wear eye protection) Turn on the N2
gas to the slowest flow possible. Purge the solution with N2 by gently bubbling with N2 gas for 2-
3 min.

15. Quickly seal the tube with the screw cap and heat to 70° C for 1 hour.

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Phospholipid Analysis via TLC

16. During this 1-hour incubation period, perform the TLC analysis of the phospholipids. Label 3-4
lanes on your TLC plate with a pencil.

17. Using a Pasteur pipette, spot a small amount of phospholipid solution onto a silica gel TLC plate
in one of the lanes.

18. Then, spot small amounts of the phospholipid standards provided into the other 2-3 lanes. Allow
the spots to dry.

19. Place the TLC plate in a small jar in the fume hood containing 5 mL of CHCl3/CH3OH/H20
(65:25:4, v/v/v), and leave it there until the solvent front is within 1 cm of the top of the plate.

20. Remove the plate from the jar and mark the solvent front with a pencil. Allow the solvent to
evaporate.

21. Visualize the sample components via iodine staining in the fume hood. Place the plate in a jar
containing a small amount of solid I2, close the jar and leave it there for about 10 min.
(CAUTION: Avoid breathing or dispersing I2.) Remove the plate from the jar and allow the
excess I2 to sublime away in the fume hood. Circle the yellowish-brown spots. The color will fade
on exposure to air, but will reappear if you put the plate back into the jar.

22. Sketch your TLC plate in your laboratory notebook. Use your sketch to identify which
phospholipids are present in your solution.

Hydrolysis of Fatty Acids from Phospholipids and Conversion to Fatty Acid Methyl Esters
(FAMEs) continued

23. Allow the sample from above to cool to room temperature. (CAUTION: Do not perform next step
until the sample is at room temperature.)

24. Add 1 mL of hexane to the solution. Mix well, then allow the two phases to separate. Pipette the
top (hexane) layer into another tube.

25. Repeat step 24 two more times, and combine the hexane fractions.

26. Reduce the volume of the combined hexane fractions to about 0.5 mL by evaporation under N2.

Analysis of FAME's by GC/Mass Spectrometry (from Synthesis Laboratory)

The Hewlett-Packard G1800B GCD System is an integrated GC/mass spectrometry system. The gas
chromatograph is based on the HP 5890 Series II gas chromatograph with an electron ionization
detector (EID). The system offers quantitative chromatographic information and qualitative tools for
rapid compound identification. Along with abundance and retention time information for each peak
in a chromatogram, the GCD gives the routine user a "third dimension" of data - mass spectra. A
mass spectrum can be automatically searched against a reference in a commercial or user-created
library for compound identification.

27. The GCD system will have been activated and tuned prior to your arrival in lab.

28. Using the mouse and appropriate menu commands, go to Acquire Data. Select "single run".

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29. You will be presented with a menu that has a place for a data file name. Fill in a name that
contains your initials, underscore and the letter "a" e.g. mep_a [a ".D" suffix will be added to the
file name]. Also, provide a response for operator name and sample name.

30. Draw 1
L of the FAME standards solution into the syringe, taking care not to draw in air
bubbles.

31. Position the arrow on Run Method and click the left button on the mouse to activate the data
acquisition mode. The system will proceed to initiate the run sequence. An acquisition menu will
appear prompting you to wait for the "not ready" light to go off.

32. When prompted, inject the 1
L sample into the gas chromatograph and push the start button on
the GC panel. After your injection, rinse the syringe well with hexane.

33. After the injection, a window will appear on the monitor asking if you desire to override the
solvent delay. Respond negatively.

34. The display will then present you with the total ion current in real time. You may adjust the
scale using the abundance arrows. However, do not adjust any other setting until after the
chromatographic run has been completed.

35. After the chromatographic run is complete, go to the Review Data section on the menu. On this
menu select File, then select Load, select your data file and click OK.

36. You should now be presented with the total ion chromatogram on the monitor. Print this out
using the File menu, click on Print and Print Selected Window. A "2" should appear in the box;
select OK.

37. After the chromatogram is printed, you may analyze your data. Use your knowledge of the
structure of each FAME and of mass spectrometry to identify each peak in your chromatogram.

38. To analyze your data, first zoom in on selected peaks in the chromatogram. Do this by moving
the arrow to the left of the peak(s) you wish to zoom in on, depress the left button of the mouse
and drag the arrow through the region you desire expanded. Note: the arrow will be replaced by
a box during the dragging step. Releasing the left button will initiate the display of the
expansion.

39. To obtain the mass spectrum of a particular component, move the arrow to the peak and double
click the RIGHT button on the mouse.

40. Verify that all peaks in the chromatogram are individual components and not unresolved
mixtures by viewing the mass spectrum of various peaks of the chromatogram. You may
accomplish this by using the mouse to toggle between your spectra.

41. Print out at least one mass spectrum for every isolated component in your chromatogram. To
restore the chromatogram or the mass spectrum to its original scale, double click the left button
of the mouse anywhere in the window.

42. The single ion chromatogram can be used to quickly identify mixture components that possess a
fragment ion in common. For example, if you desire to quickly identify mixture components that
possess a monosubstituted benzene, you may select an ion of mass 77 and display the
chromatogram of abundance of ion mass 77 as a function of time. To produce the ion

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 chromatogram, go into the Chromatogram menu and select Extract Ion Chromatogram. Fill in
the nominal masses that you desire for the Ions. The monitor should then display the
chromatogram for only the ion(s) selected.

43. Select each isolated component of the standard FAME mixture, and pull up the mass spectrum.
Then pull down the menu labeled Spectrum and select Tabulate. A table listing the mass to
charge ratios and ion abundances will be displayed on the monitor. Print these tables. Print out
the mass spectra by using the File menu, click on Print and Print Selected Window and place a
"1" in the box; select OK.

44. Repeat the Data Acquisition and Analysis for the Egg FAMEs. But in this case change the file
name to your initials underscore b, (e.g. mep_b). Select Exit in the File menu.