BIOL 305L Laboratory Four

Transcription

1 Please print Full name clearly: BIOL 305L Laboratory Four An investigation of biochemical changes to tomato fruit when it ripens Introduction According to an old country song by John Denver, the only two things money can't buy are true love and homegrown tomatoes (I am being honest here, feel free to check out the following link on youtube if you don t believe me: Oh, and PLEASE, do NOT come to me and ask who John Denver is!!!!! Shame on YOU if you don t know!!!!!!!! Anyway, the tomato has become a model system for the study of fruit ripening and, more particularly, the changes that bring about fruit softening. In this fruit, despite some results implicating the hemicellulosic and cellulosic cell wall fractions in fruit softening, the most recent work in the area has focused on the pectic fractions. In particular, there has been much interest in the polyuronidesolubilizing enzyme, polygalacturonase. Its activity rises dramatically during ripening, and a close correlation between polygalacturonase and pericarp softening has been demonstrated in a range of tomato cultivars. Naturally, tomatoes unevenly ripen, showing darker green patches when unripe and variable redness when ripe -- traits that still show up in garden-variety and heirloom breeds. However, in the late 1920s, commercial breeders stumbled across a natural mutation that caused tomatoes to ripen uniformly -- from an even shade of light green to an even shade of red. This "uniform ripening" mutation has become indispensable to the $2 billion a year U.S. commercial tomato market, showing up in almost all tomatoes produced for grocery stores. The uniform redness makes it ideal for groceries, where customers expect evenly colored, red fruit. An important fact to remember: Whilst leaves are the primary photosynthesis factories in a plant, developing tomato fruit can contribute up to 20 percent of their own photosynthesis, yielding high sugar and nutrient levels in fully ripe fruit. The uniform ripening mutation, which commercial breeders select for, eliminates this protein in the fruit, therefore reducing sugar levels and nutrients in the fruit.

2 So, how can you look at biochemical alterations to tomato fruit as they ripen during a three hour lab? The act of ripening requires the expression and action of an enzyme protein to break down structural cell wall polysaccharides to smaller units and monosaccharides. So there should be a change in both sugar content and protein content between ripe and unripe tomatoes, right? So, write your hypothesis here: Biochemical assays to be used in this Laboratory Benedict's reagent assay procedure for reducing sugars: This reagent contains 100 g sodium carbonate and 173 g sodium citrate dihydrate in a final volume of 850 ml water. Slowly, with stirring, add a solution of 17.3 g copper sulfate pentahydrate in 100 ml of water. Bring the final volume to one liter. The blue copper (II) ions from copper (II) sulfate are reduced to red copper (I) ions by the aldehyde groups in the reducing sugars. This accounts for the color changes observed. The red copper (I) oxide formed is insoluble in water and is precipitated out of solution. This accounts for the precipitate formed. As the concentration of reducing sugar increases, the nearer the final color is to brick-red and the greater the precipitate formed. Sodium carbonate provides the alkaline conditions which are required for the redox reaction above. Sodium citrate complexes with the copper (II) ions so that they do not deteriorate to copper (I) ions during storage.

3 Procedure: Clearly label clean test tubes for your standards. Use a 2% glucose solution and distilled water to prepare a series of standard glucose solutions of different concentrations: 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, 1.2% 1.4%, 1.6%. 1.8%, and 2% Add 5ml of each standard and 1 ml of Benedict s solution together. Mix the contents of each tube by gently shaking the test tubes back and forth. Place the tubes in a test tube rack and set the rack in the BOILING water bath. CAUTION! The water is very hot. Incubate the tubes for 20 minutes. Remove your test tubes and allow them to cool. Transfer the contents to 1.5 ml centrifuge tubes and spin for two minutes. If there are still suspended debris in any of the tubes, centrifuge for two more minutes. **It is important that the solution be clear for the absorbance measurements. If there is solid matter suspended in the solution, the light being sent through the sample will be scattered and will cause error in the measurements. Decant the supernatants into clean, labeled test tubes. Be careful that any sediment remains in the pellet at the bottom of the centifuge. Determine the O.D. of each of the samples using a spectrophotometer set to read at 735 nm. Use distilled water to set the 100%T. Plot the average values in Excel.

4 Lasker and Enkelwitz assay procedure for Ketones (ie fructose): The Lasker and Enkelwitz test also utilizes Benedict's solution, although the reaction is carried out at a much lower temperature. The color changes that are seen during this test are the same as with Benedict's solution. In this assay, samples are heated in a 55 o C water bath for minutes. Ketopentoses demonstrate a positive reaction within 10 minutes, while ketohexoses take about 20 minutes to react. Use a 1% fructose solution and distilled water to prepare a series of standard fructose solutions of different concentrations: 0.05%,0.075% 0.1%, 0.2%, 0.4%, 0.6%, 0.8%, and 1% Add 5ml of each standard and 1 ml of Benedict s solution together. Mix the contents of each tube by gently shaking the test tubes back and forth. Place the tubes in a test tube rack and set the rack in the 55 o C water bath. CAUTION! The water is very hot. Incubate the tubes for 10 minutes. Remove a sample for centrifugation. Replace the rest of the sample back in the 55 o C water bath. Remove your test tubes and allow them to cool. Transfer the contents to 1.5 ml centrifuge tubes and spin for two minutes. If there are still suspended debris in any of the tubes, centrifuge for two more minutes. **It is important that the solution be clear for the absorbance measurements. If there is solid matter suspended in the solution, the light being sent through the sample will be scattered and will cause error in the measurements. Decant the supernatants into clean, labeled test tubes. Be careful that any sediment remains in the pellet at the bottom of the centifuge. Determine the O.D. of each of the samples using a spectrophotometer set to read at 735 nm. Use distilled water to set the 100%T. Plot the average values in Excel.

5 Bradford assay Procedure: The Bradford assay is a very popular protein assay method because it is simple, rapid, inexpensive and sensitive. The Bradford assay works by the action of Coomassie brilliant blue G-250 dye, which specifically binds to proteins at arginine, tryptophan, tyrosine, histidine and phenylalanine residues. Advantages Fast and inexpensive Highly specific for protein Very sensitive Compatible with a wide range of substances Extinction co-efficient for the dye-protein complex is stable over 10 orders of magnitude (assessed in albumin) Dye reagent is complex is stable for approximately one hour. Disadvantages Non-linear standard curve over wide ranges Response to different proteins can vary widely, choice of standard is very important Procedure: Add 1.4 ml of 1X Bradford reagent in each microfuge tube. Into the standard tubes place the following amounts of BSA protein standard (1 mg/ml). Tube µl BSA µl H 2O µg protein added Determine the absorbance at 595nm Plot the graph and find the linear regression line for the standard curve.

6 Objectives for week one: During this lab, you will build detailed standard curves for each of the three assays to be used, with at least three repeats for each standard curve. In your groups discuss your hypothesis. For the next lab: As a group bring a copy of each standard curve to include error bars for each value on the standard curves. As individuals, I expect you all to type up at least a page to explain the biochemistry behind tomato fruit ripening. Include at least three scientific papers as references. Objectives for week two: Extract saccharides and proteins from ripe and unripe tomato fruits. This may be done for you, but the rapid extraction procedure to be used is a s follows: Fruits from each plant were ground using a blender and the fruit homogenate (40g) was added to 70 ml of 80% methanol, boiled for 15min (with a loose-fitting cover), cooled, and strained through two layers of cheese cloth. Carry out all of the assays and collect data. Repeat each assay at least five times For the week after Exam two: Individual 9-10 page write up of the lab section (1 inch margin, 12 pt font, 1 ½ spacing). You know what to include! Just in case: Intro with background info and hypothesis: at least three references Methods in detail Written description of data, including standard curves. Legends and titles, and paste graphs into word! Detailed conclusion, explain results, include info from introduction. Did the experiment work? Possible errors and/or improvements. All references listed. No required format for this just be consistent!