Tarai

FIGURE 7.15 Illustration of taxol molecule and the three-ion series observed following fast atom bombardment (FAB) mass spectrometry of taxol. (From McClure, T. D., Schram, K. H., and Reiner, M. L. J., J. Am. Mass Spectrom., 3, 672, l992. With permission.)

7.4.2 Other Methods

In addition to chromatography and MS, chemists and molecular biologists employ other ways to characterize and identify plant metabolites (Figures 7.16 and 7.17).

• Nuclear magnetic resonance (NMR) — This is a spectroscopic technique used to characterize the nuclear properties of elements, proton chemical shifts in substituents on molecules or protons associated with double bonds in cyclic and noncyclic hydrocarbons, and chemical shifts of isotopes (e.g., carbon, nitrogen, phosphorous, boron, silicon, and fluorine).

• Infrared (IR) spectroscopy — This is also a spectroscopic technique that involves measurement of the absorption spectrum of a given molecule in the infrared portion (wavelengths longer than those of visible light, occurring above the red end of the light spectrum) of the electromagnetic spectrum. Every molecule has its own characteristic IR spectrum. It refers to the absorption frequencies of single bonds to hydrogen, triple bonds, cumulated double bonds, carbonyl bonds, aromatic bands, and miscellaneous bands for such molecules.12

• Melting point — Every compound has its own characteristic melting point (the temperature, in degrees Celsius, at which a solid melts). A good place to find this kind of information is in The Handbook of Chemistry and Physics.13

FIGURE 7.16 Dr. Soo Chul Chang performing an electroelution of a protein for subsequent amino analysis in Peter Kaufman's laboratory at the University of Michigan.

• Ultraviolet (UV) spectrum — This is still another spectroscopic technique that refers to the absorption spectrum for a given molecule in the UV portion of the electromagnetic spectrum (wavelengths shorter than those in the visible light portion of the spectrum and longer than those for X-rays). Every molecule has its own characteristic UV spectrum. In practical terms, it is a good idea to have an HPLC fitted with both visible and UV light sources because in HPLC analyses, we often measure the absorbance of a given compound at wavelengths that occur either in the visible or in the UV.

• Specialized MS devices — These include tandem MS, quadripole MS, and electrospray MS devices.14 An electrospray MS is very useful for doing mass spectra for large molecules like azadirachtin from the neem tree (Azadirachta indica) and taxol from yews (Taxus spp.).

• SDS/PAGE — This refers to sodium dodecyl sulfate polyacrylamide gel electrophoresis. It is a method that allows one to separate proteins

FIGURE 7.17 Hitachi amino-acid analyzer in laboratory of Dr. Akira Okubo at the University of Tokyo.

on a gel on the basis of molecular size [in 1-D (one-dimensional) gels] and their isoelectric points [pH values where they have no net charge in 2-D (two-dimensional gels)].

• Protein sequencing — This technique allows one to determine the amino acid sequence of a given protein from its amino terminal end to its carboxy terminal end. Such information is helpful in determining the deduced nucleotide sequence of the specific DNA that makes this protein via transcription and translation. It also allows one to identify particular DNA binding domains, active sites (in the case of enzymes), and targeting sequences that get proteins to their ultimate destination (e.g., chloroplast, mitochondrion, cell wall, vacuole) in the cell.

• Cloning genes by expression in Escherichia coli (the common colon bacterium) — This technique is used by molecular biologists to amplify the amount of a given gene (DNA), especially when it and its gene product are found in low abundance in a cell, tissue, or organ. This then allows one to do biochemistry on the protein of interest which may be, for example, a rate-limiting enzyme in a metabolic pathway that leads to the synthesis of a given metabolite of interest. In order to clone the gene in E. coli, the DNA of interest must be transferred to E. coli cells so that they, in effect, become genetically transformed. Methods for transforming E. coli cells are discussed in detail in Wu et al. l997.15

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