Determination of binding affinities of salicylic acids to SABP2 using tryptophan fluorescence

BIOL 33

Arrey Besong Enyong, zlbb26@imail.etsu.edu, Department of Chemistry, East Tennessee State University, Box 70695, Johnson City, TN 37614, Dhirendra Kumar, Department of Biological Sciences, East Tennessee State University, Box 70703, Johnson City, TN 37614, and Yu Lin Jiang, jiangy@etsu.edu, Chemistry, East Tennessee State University, Box 70695, Johnson City, TN 37614.
Currently, plant diseases continue to pose a major threat in developed and underdeveloped countries. For instance, plant disease witches broom, or black pod disease causes devastating blight to many plants, such as potatoes, coffee trees and grape trees. In addition, cassava mosaic virus also causes coffee trees to wilt in east Africa. Therefore, crop diseases are major threats to food supply, especially those countries in most need, such as African countries. A plant protein SABP2 was found to be essential in plant immune systems to control plant diseases, such as the cassava mosaic virus. This is because the corresponding protein SABP2 will generate a plant hormone, salicylic acid (SA), as an alert signal, through the hydrolysis of a volatile and cell permeable methyl salicylate (MeSA). The SA will then induce programmed cell death of part of the plant for the benefit for the rest. However, when the concentration of SA is very high, the activity of SABP2 will be low. Therefore, SA has regulatory effect in signal transduction. To test how SA is bound by SABP2, currently, tritium radioactivity is applied to measure the binding affinity of SA to SABP2; however, the operation is inconvenient. Thus, a convenient assay was developed to determine the binding affinities of SABP2 to a series of synthesized substituted salicylic acids by using tryptophan fluorescence. For instance, it was found that the tryptophan fluorescent intensity of SABP2 at 323 nm decreased by ~2-fold when SA was bound. The dissociation constant for SA was determined to be ~19 nM.