Protocol

Author

Ian Wallis

Overview

¹⁴C-labeled poly(ethylene glycol) (PEG)-binding assay for the determination of the activity of tannins in plant material

Background

There are numerous methods for measuring the concentration of tannins in plants (Hagerman 1989; Hagerman and Butler 1978) (also https://www.academia.edu/25605508/The_Tannin_Handbook), but one problem with many of them is that they do not measure how tannins will affect animals. For instance, most assays give results in terms of quebracho equivalents – a standard tannin, per gram of dry matter. Ecologists then assume that herbivores will eat less of plants with high concentrations of quebracho equivalents. This is not necessarily true. The advantage of polyethylene glycol is that it is a potent tannin-binding agent that is apparently non-toxic. Spraying a solution of PEG on tanniferous plants allows animals to eat more, suggesting that PEG binds the tannins that are important to animals (Silanikove et al. 1996a; Silanikove et al. 1997). This assay, based on that by Silanikove et al. (1996b), measures how much PEG plant material binds. We have used the method predominantly for measuring PEG-binding in leaves eaten by folivorous mammals. This method detected a difference in PEG-binding between species of the major eucalypt subgenera and we proceeded to show that species of the subgenus Eucalyptus are much better protected by tannins than are species of the subgenus Symphyomyrtus (Wallis et al. 2010). Using non-radioactive PEG is an important step in our measure of available nitrogen (DeGabriel et al. 2008)

Materials/Equipment

• Stock solution containing 100 gL^-1 PEG 4000 (Sigma, analytical grade) in 0.05M buffer Tris-BASE (Sigma), pH 7.1, spiked with 50 Ci 14C-PEG 4000 (Amersham).
• Working solution: 1 part of stock solution + 2 parts distilled water.
• Micro-centrifuge
• Screw-top tubes (e.g., Sarstedt 72.694.100) with an O-ring seal that will fit a micro-centrifuge. Do not use standard flip-top tubes.
• Autopipette and tips suitable for transferring 40 L; calibrate before using or ideally weigh the amount transferred.
• Scintillation vials for counting 4mL of fluid
• Liquid scintillation fluid (eg Insta-Gel 11, Packard)
• Bag suitable for disposal of items contaminated with radiation.
• Oven (37^∘C)
• Access to a radiation working area.
• 2 mL micro tube with a screw cap containing an O-ring (eg Sarstedt 72.694.100).
• Disposable transfer pipettes.

Units, terms, definitions

PEG

PEG-binding is expressed as g per 100g of dry plant matter.

Procedure

1. The procedure models well with near infrared reflectance spectroscopy (NIRS) so an optional first step is to scan all samples with NIRS. The advantage of this is that later samples can be predicted, which will minimize the use of radioactive material.
2. In duplicate, weigh 0.5000 ∓ 0.0100 g of eucalypt leaf (ground to pass a 1 mm sieve) into a pre-weighed 20 mL plastic scintillation vial. Note, write down the mass of the empty vial and lid.
3. In a radiation working area, add ca 7.5 mL (ca 0.125 Ci) of working solution decanted from a bottle using a calibrated plunger.
4. Cap the vial, reweigh and vortex.
5. When all samples are prepared (or every 30 min), place the tubes horizontally in a stainless-steel tray in an oven at 37^∘C. A shaking water bath is suitable too but keep in mind the risk of spreading radioactive material in case of a leak.
6. Incubate for exactly 24 h with occasional mixing. It seems sensible to start the incubation mid-morning and mix (vortex) several times that day.
7. Remove tubes in order and, in a radiation working area, decant ca 1.5 mL of the incubated sample into a pre-labelled 2 mL micro tube with a screw cap containing an O-ring (eg Sarstedt 72.694.100) using a disposable transfer pipette. Using these tubes will ensure that there is no leakage upon centrifugation.
8. Centrifuge for 15 min at 14000 rpm
9. Decant 40 L (Note: pipette needs to be calibrated beforehand or, ideally, weigh the supernatant to 0.1 mg) of the supernatant into 4 mL of scintillant (Packard Emulsifier-Safe) in a pre-labelled plastic scintillation vial. Vortex.
10. Count for 5 min or 10,000 counts.

Standard: count triplicate 40 L samples of the working solution.

Blank: count triplicate 40 L samples of buffer.

Calculation:

Where, C_st, C_bl and C_sm are the ¹⁴C counts of the standard, blank and sample, respectively. A_PEG is the amount of PEG in the test tube and Sw is the dry weight of the plant tissue. PEG-binding is expressed as g per 100g of dry plant matter.

Notes and troubleshooting tips

Silanikove (1996b) found similar PEG binding with 0.1, 1.0 and 2.0 g of sample and after drying samples at 50 and 90^∘C. Maximum binding took 8h at 40^∘C or 24 h at 20^∘C. PEG binding was maximal at pH7 and 10-20% lower at pH 5 and 9. The repeatability of the assay on a particular plant sample was high (CV 0.5% or lower). One trouble we encountered occurred with counting. Despite the manufacturer’s claims, scintillation fluids do not support much PEG and thus it is important to count small volumes. This explains why we measure the mass of supernatant that we count.

Links to resources and suppliers

Sigma

Amersham

2 mL micro tube with a screw cap containing an O-ring (Sarstedt 72.694.100)

Literature references

DeGabriel, J.L., Wallis, I.R., Moore, B.D., Foley, W.J., 2008. A simple, integrative assay to quantify nutritional quality of browses for herbivores. Oecologia 156, 107-116.

Hagerman, A.E., 1989. Chemistry of tannin-protein complexation. In: Hemingway, R.W., Karchesy, J.J. (Eds.), Chemistry and significance of condensed tannins. Plenum Press, New York.

Silanikove, N., Gilboa, N., Nir, I., Perevolotsky, A., Nitsan, Z., 1996a. Effect of a daily supplementation of polyethylene glycol on intake and digestion of tannin-containing leaves (Quercus calliprinos, Pistacia lentiscus, and Ceratonia siliqua) by goats. Journal of Agricultural & Food Chemistry 44, 199-205.

Silanikove, N., Gilboa, N., Nitsan, Z., 1997. Interactions among tannins, supplementation and polyethylene glycol in goats given oak leaves: Effects on digestion and food intake. Animal Science 64, 479-483.

Silanikove, N., Shinder, D., Gilboa, N., Eyal, M., Nitsan, Z., 1996b. Binding of poly(ethylene glycol) to samples of forage plants as an assay of tannins and their negative effects on ruminal degradation. Journal of Agricultural & Food Chemistry 44, 3230-3234.

Wallis IR, Nicolle D and Foley WJ (2010) Available and not total nitrogen in leaves explains key chemical differences between the eucalypt subgenera. Forest Ecology and Management 260:814-821.

AOAC – Association of Official Agricultural Chemists (The AOAC publishes a journal on official methods of chemical analysis)

Health, safety & hazardous waste disposal considerations

General: The use of materials that emit ionizing radiation requires suitable facilities, training of personnel and awareness of local regulations.

Disposal of waste: Each incubation tube contains about 0.125 Ci of ¹⁴C-PEG. Although a small amount of contaminated waste (gloves, tissues) will be generated when adding the ¹⁴C-working solution to the plant material, most waste is generated at the time of preparing samples for counting. For instance, the volume incubated that is not centrifuged (the plant material and about 5.5 mL of working solution) contains ca 0.09 Ci while the volume centrifuged that is not counted, contains about 0.03 Ci of ¹⁴C-PEG. Both sets of vials and their contents will be disposed together. A minimal amount of the original working solution is actually counted. The vials containing the scintillant and sample will be disposed together. The method of disposal depends on local regulations.