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The Influence of pH on the Color of and

RandyMoore DarrellS. Vodopich

Anthocyanins are a water-soluble Downloaded from http://online.ucpress.edu/abt/article-pdf/49/2/111/42721/4448451.pdf by guest on 03 October 2021 group of pigments called that are widespread among angio- sperms, usually absent in liverworts and algae, and rare in mosses and gymnosperms. More than one antho- Figure 1. Structureof .Position 3 of ring A is-always glycosylated with glu- cyanin often occurs in the same flower cose galactose, rhamnose, xylose-glucose, rhamnose-glucose,or glucose-glucose.Position 5 or organ, and these pigments can im- is sometimes glycosylated-if so, it is glycosylatedwith glucose. Position 7 is almost never glucose. part violet, blue, purple, red and glycosylated-if so, it is glycosylated with scarlet colors to flowers, stems, fruits and leaves. pigments called betacyanins and be- Materials present taxanthins. Despite their similar Anthocyanins are typically 10-15 ml capacity or two sugar mol- colors, betalains are unrelated to an- 10 test tubes, as glycosides of one cabbage, 20 g to anthocyanidin thocyanins, and are found in only 10 red ecules attached red beet roots, 20 g 1), which is also colored. The families of plants, all of which are (Figure balance to weigh tissue presence of sugars on the molecule in- members of the order Caryophyllales and allows an- (Centrospermae). Indeed, betalains blender creases its solubility, buchner funnel thocyanins to accumulate in the and anthocyanins do not occur in the vacuole of same plant-individual plants cannot filter paper aqueous sap of the central sidearm flask, 500 ml capacity plant cells. Hydroxylation or methyl- synthesize both of these pigments. Betalains, like anthocyanins, are water aspirator ation of ring B of anthocyanidin 2 beakers, 500 ml capacity, with (Figure 1) produces anthocyanins of glycosides of sugars and colored groups. For example, (the be- labels different colors (Figure 2). N HCI, 20 ml function in talain in roots of red beets) is a glyco- 0.1 Anthocyanins probably 0.01 N KOH, 20 ml attracting pollinators to flowers, and side of glucose and betanidin. Beta- also contribute to the spectacular nidin is red and has the structure colors of autumn leaves. Yellow and shown in Figure 3. Like anthocyanins, orange (e.g., in tomatoes) betalains are water soluble and accu- responsible for similar colors are unre- mulate in the vacuole of plant cells. Randy Moore is an associate professor of lated to anthocyanins. Anthocyanins and betalains can be biology at Baylor Univ., Waco, Texas, The color of anthocyanins depends distinguished by their differing re- 76798. He received his B.S. in Biology from on several factors: sponses to changes in pH-betalains Texas A&M Univ., M.S. in Botany from the typically do not undergo extensive Univ. of Georgia and Ph.D. in Biology Associationswith other compounds. For changes of color with pH as antho- from U.C.L.A. His professional member- for example, an association with phe- cyanins do. The following experiment ships include the American Society & Space Biology, Botanical nolic compounds usually produces rapid and inexpensive Gravitational is a "sure-fire," Society of America, American Society of a blue color. means of demonstrating the influence Substituentgroups on the B ring of Plant Physiologists, and American Society of pH on the color of anthocyanins interests in- For example, the for Cell Biology. His research anthocyanidin. and betalains. how plants perceive and on this clude determining presence of methyl groups respond to gravity. Darrell Vodopich is an ring reddens the pigment. assistant professor of biology at Baylor pH of the cellular sap. Most antho- Objectives Univ., Waco, Texas, 76798. He received his cyanins are purple or blue at high The objectives of this experiment B.S. and M.A. in Biology from East Caro- in from pH, and become reddish as the pH are to demonstrate 1) the effect of lina Univ. and his Ph.D. Biology of South Florida. His professional decreases. This pH-dependent pH on the color of anthocyanins Univ. in color results from ioniza- memberships include the North American change and betalains, and 2) how a So- tion of hydroxyl groups on the B Benthological Society and American simple chemical test (i.e., His research in- ring, followed by electron shifts on ciety of Limnologists. changing pH) can be used to dis- terests include the ecology of freshwater the ring. tinguish between betalains and invertebrates, and predation in aquatic en- Betalains include red and yellow. anthocyanins. vironments.

INFLUENCEOF pH ON COLORS 111 OH (crimson) (purple) Figure 2. Cyanidin (first isolated from blue cornflower)is a hydroxylatedanthocyanidin, whereas petunidin (isolated from petunias) is a

methylated anthocyanidin. Downloaded from http://online.ucpress.edu/abt/article-pdf/49/2/111/42721/4448451.pdf by guest on 03 October 2021

0.1 N KOH, 20 ml This can be demonstratedby adding Can differences in pH account for crystalline KOH or NaOH, 20 drops of 0.1 N HCI to tubes 3, 4, 8 all blue or-red colors of flowers con- pellets and 9, or 1 N HCl for tubes 5 & 10. taining anthocyanins? Why or why 1 N HCl, 30 ml Observe the changes in color. not? test tube rack Questions for Students: Procedure Do betalains and anthocyanins FurtherReading 1. Separately homogenize 20 g of show the same changes in color in re- Goodwin, T.W. (Ed.) (1976). Chemistryand red cabbage and red beet roots in sponse to differingpH values? Why or biochemistryof plant pigments.(2nd ed.). 400 ml of water in a blender. why not? Vol. 1. London: Academic Press. 2. Filter each homogenate through filter paper. This filtration is facili- tated by using a buchner funnel in a sidearm flask attached to an aspi- rator. 3. Discard the filter and solid mate- rial. 4. Place the two extracts into sepa- rate labeled flasks. 0000~OO 5. Add 5 ml of cabbage extract con- taining to each of 5 test tubes numbered 1-5. 6. Add 5 ml of beet extract con- taining, betalains to each of 5 test tubes numbered 6-10. 7. Perform the following treatments: Tubes 1 & 6: None (untreated controls) Tubes 2 & 7: Add 1.0 ml of 0.1 N HCl, mix, and note any color change I Tubes3&8: Add 0.5 ml of 0.01 N )~~~~~ KOH, mix, and note any color change (the cabbage extract will become violet or, if too basic, blue) Tubes 4 & 9: Add 1.0 ml of 0.1 N KOH, mix, and note any color change HOOC N COOH Tubes 5 & 10: Add one pellet of KOH or NaOH, mix, and note any color change (the antho- cyanin will become yellow) 8. These pH-dependent changes in H pigment color are fully reversible. Figure 3. Structure of betanidin.

112 THE AMERICAN BIOLOGY TEACHER, VOLUME 49, NO. 2, FEBRUARY 1987