In the Laboratory
Determination of the Fatty Acid Content of Biological Membranes: A Highly Versatile GC–MS Experiment
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Emeric Schultz* and Michael Eugene Pugh Department of Chemistry, Bloomsburg University, Bloomsburg, PA 17815-1301; *
[email protected] Introduction and Background Chemical education is changing dramatically. Growing emphasis is being placed on content that “connects” with the career goals of the majority of students in the first two years of chemistry (e.g., bioscience majors); on experimental experiences that involve discovery and cooperative learning; and on experiments that incorporate modern instrumentation, especially instrumentation that will be encountered in the workplace. In this paper we describe a highly versatile experiment that addresses all of these concerns. From the perspective of biochemistry, which we both teach and which is at the “end of the chemistry trail” for many students in chemistry course sequences, there are certain concepts that we feel warrant thorough development. Perhaps the most important is understanding the nature of weak interactions. The integrity of membranes and DNA and the manner in which proteins assume their final shape (and thus their functional potential) are among the key places where weak interactions are important. Ideally, earlier course coverage, preferably with lab emphasis, would put in place concepts that could be fully developed in a chemistry-focused biochemistry course. The experiment described here has this potential. Both gas chromatography and mass spectrometry are important techniques with which all students should gain experience in chemistry sequences. The combined technique (GC–MS) is one of the key instrumental techniques used today. Several experiments that use GC–MS and involve biomolecules have been reported in this Journal. These typically involve the characterization of fatty acids in fats and oils from plant and animal sources as their fatty acid methyl ester derivatives (FAMEs) (1–4 ) or the identification of amino acids in peptides (5, 6 ). Overview and Experimental Details The experiment described in this paper, which is done in two different places in our curriculum, involves the GC–MS of FAMEs obtained from bacterial membranes. Frontiers in Science and Technology is a non-science-majors lab course that has previously been described in this Journal (7 ). It has been a part of the honors program at Bloomsburg University since 1996 and is taken primarily by freshman and sophomores. We are modifying this course to be used as a handson science course for elementary and middle school education majors. Biochemistry is a junior/senior level course taught from the chemistry perspective and is a requirement for all B.S. biology majors; it also meets the ACS requirement for all chemistry majors. In both courses bacterial cells are grown. To a chemist unfamiliar or unwilling to deal with microbiology, this may be offsetting. In fact, neither of us, although we were trained as biochemists, knew how to grow bacterial cells until recently. 944
Centrifuge cell culture & pour off supernatant
Resuspend in 2.5 mL methanol:toluene (4:1) & transfer to screw cap tube
Add small stirring bar & 200 µL of acetyl chloride; cap securely and heat in boiling water bath for 60 minutes
Centrifuge; remove top layer; inject 1 µL into GC/MS
Neutralize with Na2CO3 Transfer to Corex centrifuge tube and add 1.0 mL toluene & mix
Figure 1. Protocol for preparation of fatty acid methyl esters.
We were forced to learn in order to keep up with trends in biochemistry, especially the biotechnology revolution. It is not that hard! In fact the preparation for this experiment is far less intricate (and smelly) than the preparation for certain general and organic labs done in this department. The steps from cell cultures to the analysis of FAMEs by GC–MS are outlined in Figure 1. Beginning with the suspended cells, the time to injection is about 2 h. If students start with pelleted cells, the time is cut to about 11⁄2 h. The only part of the experiment that may cause concern is the addition of acetyl chloride, which acts as a catalyst for transesterification of esterified fatty acids in phospholipids and sphingomyelins (8). The instructor adds this slowly via syringe to each student preparation. The only other concern is that if the reaction tubes are not well sealed, solvent does evaporate during the course of the heating. Replenishment of methanol/toluene addresses this problem. Our experience over the last 5 years has been that with rare exceptions (i) students get more than enough FAMEs to be detected by GC–MS; (ii) preparations are exceedingly clean—there are few, if any, contaminants in the GC profile where FAMEs are found; and (iii) the results are highly reproducible among student groups and over the years (for the same growth conditions). The Experiment in a Course for Nonscience Majors In the Frontiers course our goals for this experiment are fairly limited but fit in nicely with other aspects of the course. The major portion of the experimentation in the Frontiers course is the cloning of the gene for bioluminescence from one bacterium (Vibrio fischeri) to another (Escherichia coli). We grow the E. coli clone containing the plasmid for bioluminescence isolated by each student team; in addition we grow “standard” V. fischeri and E. coli. Each student team obtains FAMEs from its clone and one of the standard bacteria; the other standard is available by sharing. Students are asked to
Journal of Chemical Education • Vol. 78 No. 7 July 2001 • JChemEd.chem.wisc.edu
In the Laboratory Table 1. Versatility of GC–MS of Membrane FAMEs Course
Experimental Determinations
Learning Goals
Student Output (Reports)
Non-science- GC profiles of bacterial membranes; majors lab MS of one of GC components course
Isolation, separation, and characterization of a family of substances; chemical fingerprinting; molecular weight determination
Identification of a bacterium using GC–MS; determining the presence of a specific bacterium or a bacterial mixture (identifying an “unknown”); how separations can be done
General chemistry
GC profiles of bacterial membranes; MS of several GC components; molecular weights
Separation and rationale for separation of a mixture into its components; molecular weight determination from molecular ions; isotopic abundance (qualitative); cumulative strength of weak attractions
How separations can be done; evaluating retention time vs chemical structure and importance of weak interactions
Analytical chemistry
GC profiles of bacterial membranes; MS of several GC components; molecular weights; isotopic abundance (quantitative)
Theory and practice of GC, MS, and combined technique; molecular weight determination from molecular ions; high-resolution determination of chemical formulas
Separations and factors affecting separations using GC (column and instrument parameters); information available from MS; interpretation of MS information
Organic chemistry
GC profiles of bacterial membranes; Ester synthesis; chromatographic separation of a Synthesis and characterization; identification of an MS of several GC components; molec- mixture; fragmentation patterns and connectivity; “unknown mixture” (bacterial membrane); identificaular weights; fragmentation patterns determination of molecular weight tion by fragmentation pattern and molecular ion
Biochemistry GC profiles of bacterial membranes; MS of GC components; quantitative differences in membrane FAMEs
Derivatization and analysis of a major biochemical family; cumulative strength of weak attractions and correlation to membrane fluidity
compare the GC–MS fingerprint of their clone to those of the standards. There is an additional bit of chemistry learning that we do using the computer library and mass spectral fragmentation matches. The library identifies each peak by name (e.g., dodecanoic acid methyl ester) and gives a confidence level to the assignment (confidence levels for the common FAMEs are usually 95% and higher). By this point in the course students have some familiarity with rudimentary chemical structures. Commonalties in the names (all are acids; the common decanoic has different prefixes) lead to certain generalizations. Once the common structural base (hydrocarbon chain and methyl ester group) is present, students “play” with getting the pieces seen in a mass fragmentation pattern for a given substance. All you have to be able to do is add combinations of 1, 12, and 16 (the whole-number mass values for H, C, and O, respectively). This exercise although informal, puts some scientific flesh onto the bare-boned notion of each substance having a distinct fingerprint. The Experiment in Junior/Senior-Level Biochemistry In the biochemistry course the emphasis is quite different. The nature of membranes is a major topic in a typical biochemistry course (or textbook). Our coverage of membranes connects to the major emphasis in our biochemistry course, the importance of weak interactions (enthalpy effects) and entropy in determining biological structure. By this point in the course the final native structures of proteins and carbohydrates have been thoroughly investigated. For lipids we start from the concept of homoviscous adaptation, the ability of microbes and some other organisms to alter the composition of their membranes in response to changing environmental conditions (9). We grow bacterial cultures at two temperatures (for E. coli, ca. 22 °C and 37 °C). Research teams of 3 students obtain two FAME samples ready for GC–MS after about 2 h. The students can start the analysis of their FAME samples in the same lab period or after the period has ended. (We assign students to research groups partly on the basis of their schedules, and there is always at least one team member who can
Identification of a bacterium using GC–MS; evaluating types and quantities of different chemical structures and cumulative strength of weak interactions
perform this task.) The results verify the standard textbook statements that the ratio of saturated to unsaturated fatty acids increases with an increase in growth temperature (10, 11). There is also a substantial increase in the appearance of oddchain-length fatty acids. We set the computer printout to give a copy of the GC profile and summary MS report for each sample. The summary MS lists the 3 best computer library matches (by IUPAC name) for each GC peak. Students are instructed to select and organize the data output. We intercalate a DNA isolation using the major portion of cells grown at 37 °C (FAME analysis requires only 10 mL of cell culture). This keeps all members of the group busy with one task or another, decreases dead-time significantly, and provides a high-density laboratory experience (7). One of the experiments we do with the isolated DNA measures the hyperchromatic shift. As the DNA solution is heated, the two chains come apart as hydrogen bonds are broken; the absorbance increases as the bases become more exposed; after the solution is allowed to cool, the DNA reanneals and the absorbance decreases. These two experiments wonderfully complement each other and clearly demonstrate how the disruptive force (heat) affects the cohesive forces (weak interactions) holding two very different types of biomolecules together. A team report comparing and contrasting the reasons for the integrity of DNA and membranes completes the combined experiments. Applications and Utility of the Experiment How this experiment could be used in various chemistry courses is summarized in Table 1. The items for each course were identified from conversations with colleagues and are certainly not exhaustive. It is interesting to consider how this experiment could be done in a lower-level course with certain objectives in mind and then be revisited in a higher-level course with other objectives. We are currently discussing that idea. The “wild card” is whether and to what extent the computer library should be used. This would depend upon the specific learning objectives. For instance, if one wants students to figure out fragmentation patterns and assign structures, the computer library could be turned off.
JChemEd.chem.wisc.edu • Vol. 78 No. 7 July 2001 • Journal of Chemical Education
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In the Laboratory
In summary, this experiment should appeal to biology majors, can be used to develop several important chemistry concepts, involves teamwork, employs an important major instrument, and can exploit computer power to whatever extent desired. In addition, departments seeking to fulfil the new ACS biochemistry requirement could put this experiment into the laboratory portion of a course other than biochemistry. Hazards The use of acetyl chloride may present some problems for certain audiences. Instructors can easily add this substance to the samples. Good practice in working with bacterial cultures is to pour unused cell fractions and spent cultures into a disinfectant. Clorox works nicely. W
Supplemental Material
Supplemental material for this article is available in this issue of JCE Online.
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Journal of Chemical Education • Vol. 78 No. 7 July 2001 • JChemEd.chem.wisc.edu
