Plant Physiology (Biology 327)  - Dr. Stephen G. Saupe;  College of St. Benedict/ St. John's University;  Biology Department; Collegeville, MN  56321; (320) 363 - 2782; (320) 363 - 3202, fax;    ssaupe@csbsju.edu

Measuring Anthocyanin Content in Isolated Red Cabbage Protoplasts

Background
:
In this exercise we will spectrophotometrically determine the anthocyanin content of the red cabbage protoplasts in an acidified methanol extract. This calculation is based on the Beer-Lambert Law: A = ecl [where e = molar extinction coefficient of anthocyanin which is 38,000 M-1cm-1 (Hrazdina et al, 1982); c = concentration in M; l = pathlength in cm, which is usually 1.0 cm (or 1.2 cm in a Spec 20)].  For more on measuring anthocyanin concentrations, click here.

Questions: What is the anthocyanin content of a red cabbage protoplast? Is the concentration of anthocyanin similar to that of red grape protoplasts (1.67 pmol/protoplast, 0.097 M; Moskowitz and Hrazdina, 1981)?

Hypothesis: The anthocyanin content of red cabbage will be approximately 2.0 pmol/protoplast or 100 mM.

Protocol:

1. Transfer 0.1 ml aliquots of the partially purified protoplasts to each of five small tubes containing 0.9 mL of acidified methanol (1% conc. HCL in MeOH).  Be careful working with this solution. You must wear goggles and gloves when handling. Stopper and gently vortex.  Centrifuge to clarify the solutions if necessary.

2. Record the absorbance of the resultant solutions at 553 nm against a blank of acidified methanol (Table 3).

Data:

1. Complete Tables 1 and 2.
 Table 1: Absorbance and anthocyanin content in a 0.1 ml sample of a partially purified red cabbage protoplast suspension in acidified methanol. Sample A553 [Anthocyanin] (M) (in cuvette) [Anthocyanin] (M) in sample (remember dilution) [Anthocyanin] (pmol/protoplast) [Anthocyanin] in a protoplast (M) 1 2 3 4 5 6 7 8 9 10 Mean (+/- std dev)

 Table 2: Summary Table Mean Protoplast Volume (pl) (from our ocular micrometer data; previous lab) Mean [protoplast] (protoplasts ml-1; hemocytometer data) Mean [anthocyanin] (pmol/protoplast) Mean [anthocyanin] (M)

Analysis and Conclusions:

1. Calculate the anthocyanin concentration (Table 2). Don't forget to include the dilution factor in your calculation.
2. Calculate the anthocyanin content (pmol � protoplast–1 ) for the red cabbage leaf protoplasts. (Hint: the units of molarity are moles .liter–1 ). Show your work.
3. Calculate the molar concentration of anthocyanin (M) per protoplast. Show your work.
4. How do your data compare to published data for grape?
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Last updated:  01/07/2009     � Copyright  by SG Saupe