Team:Utah State/Results/Chlorophylasse

Mechanism

Chlorophyll is a photopigment found in the chloroplast of green plants. Chlorophyll absorbs light strongly in the blue and red regions of the visible spectrum. It absorbs very poorly in the green region, and that light is reflected, hence, making plants green. Chlorophyll is an excellent photoreceptor because it contains networks of alternating single and double bonds. Because, the electrons are not held tightly to a particular atom and can resonate, they can be readily excited by light. There are two varieties of chlorophyll molecules, chlorophyll a and chlorophyll b. They have small differences in absorbance spectrums allowing the plant to absorb and utilize more of the sun’s energy. The power of chlorophyll is its ability, when excited, to transfer an electron to an acceptor creating a separation of charge and chemical potential. Thus, light energy is converted into chemical energy (Tymoczko et. al., 2010).

Protein Gels

Chlorophyll is naturally degraded during normal turnover of the pigment, when leaves change colors during fall, when fruit ripens, and during triggered cell death due to extreme temperature or water shortage. The first step in the breakdown of chlorophyll is catalyzed by the enzyme chlorophyllase. Chlorophyll is broken down into chlorophyllide and phytol. Chlorophyll is a dark green and hydrophobic molecule while chlorophyllide is a lighter green and hydrophilic (Arkus et. al, 2007). The chlorophyllase enzyme obtained for our project was cloned from a species of wheat, Triticum aestivum. Chlorophyllide is further broken down by a series of enzyme-catalyzed reactions into colorless final products (Eckhardt et. al, 2004).

Green grass stains are actually chlorophyll stains. When a grass stain occurs the friction caused from sliding the plant over the material breaks cell membranes. This releases chlorophyll and other proteins into the fabric. Because chlorophyll is similar in chemical structure to natural fibers like cotton and wool it binds to the fabric making it difficult to remove with regular detergents (Rodriguez, 2003). Our project aims to synthetically produce the enzyme chlorophyllase that will begin the degradation of chlorophyll. As chlorophyll is degraded into chlorophyllide, it will become more water soluble. The green, water soluble, stain will then be easier to remove with normal washings.

Assays

Chlorophyll Extraction From Spinach Leaves

Chlorophyll was extracted from spinach leaves using a modified version of the one found in Arkus et. al., 2008. Six spinach leaves were ground with a mortar and pestle. Fifteen ml of chloroform:methanol (1:2 v/v) was added to the mixture and incubated at room temperature for 30 minutes. The solution was then passed through a cheesecloth into a glass beaker. Three ml’s of the solution was transferred to another glass bottle and 1 ml of chloroform with 1.8 ml of ddH2O were added. The vial was shaken to mix and centrifuged to separate phases. The upper aqueous phase was discarded and the lower organic phase was transferred to a new tube. Two to three drops of ethanol were added to clarify solution and allowed to dry out in fume hood overnight. The chlorophyll was resolubilized in 1 ml of acetone. Concentration of chlorophyll was found using a UV-vis spectrophotometer at a 1:100 dilution. Absorbances were read at 663 nm and 646 nm. The concentration of chlorophyll in the dilution equals 0.01776 X A646nm + 0.00734 X A663nm. Molarity was calculated using the molecular weight of chlorophyll, 839.5 g mol-1. The stock was stored in a -20 oC freezer.

We extracted 43.14 ± 8.93 uM of chlorophyll from six spinach leaves that was later used to test the activity of our produced chlorophyllase.

Chlorophyllase was tested using a chlorophyllase activity assay. Chlorophyll A in acetone, 0.14 mM, was mixed with MOPSO buffer, 20% v/v. Purified protein, 0.1mg, was added in a 1.0 ml reaction volume at 25 C and shaken by hand for 10 min. The reaction was quenched with a 4:6:1 (v/v/v) acetone:heptane:KOH (1 ml). After mixing the reaction tubes were centrifuged for 10 min. and 2,500g to separate phases. The aqueous phase containing the chlorophyllide reaction product was recovered and absorbance measured at 667 nm using a UV-vis spectrophotometer. Using an extinction coefficient of 76.79 mM-1 cm-1 was used to determine chlorophyllide concentrations (Arkus et. al., 2008).

The results showed an increase of 3.527 uM in concentration of chlorophyllide over the control. If the reaction runs well enough, a visual may be seen of the green color changing locations from the organic phase to the aqueous phase, after centrifugation, because of the cleaving of the phytol tail.

Future Applications

References

Tymoczko, J., Ber. J., Stryer, L. (2010) Biochemistry a short course. Ahr, K. (Ed.). New York, NY: W.H. Freeman and Company

Rodriguez, M (2003). Difficulty of grass stain removal. Message posted to http://www.newton.dep.anl.gov/ askasci/gen01/gen01433.htm

Arkus, K. A., Jez, J. M. (2008). An integrated protein chemistry laboratory. Biochemistry and Molecular Biology Education, 36(2), 125-128.

Eckhardt, U., Grimm, B., & Hörtensteiner, S. (2004). Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant molecular biology, 56(1), 1-14.

Tymoczko, J., Ber. J., Stryer, L. (2010) Biochemistry a short course. Ahr, K. (Ed.). New York, NY: W.H. Freeman and Company

Rodriguez, M (2003). Difficulty of grass stain removal. Message posted to http://www.newton.dep.anl.gov/askasci/gen01/gen01433.htm

Arkus, K. A., Jez, J. M. (2008). An integrated protein chemistry laboratory.Biochemistry and Molecular Biology Education, 36(2), 125-128.

Eckhardt, U., Grimm, B., & Hörtensteiner, S. (2004). Recent advances in chlorophyll biosynthesis and breakdown in higher plants. Plant molecular biology, 56(1), 1-14.