Our preliminary studies suggest that in water, leaf leachates of Amur Honeysuckle (Lonicera maackii) decrease dissolved oxygen to hypoxic levels and reduced survivorship of developing anuran larvae. This finding indicates a high likelihood of an oxygen-binding particle within L. maackii leaves or as a result of their decomposition. To determine the agent responsible, teas were made from leaves of both L. maackii and native riparian trees. Teas were then spectrophotometrically assayed (Prussian Blue test) for plant phenolics (e.g., tannic acid) as a potential factor responsible for oxygen loss. These analyses were completed in fresh leaf material, and in samples at various stages of aquatic leaf decomposition. When subjected to boiling temperature or low pH acid conditions, the teas returned to normal dissolved oxygen levels after aeration. A series of tests were then used to determine if a potential plant protein was responsible for binding oxygen. Dialysis filtrations (3500 Dalton) of teas eliminating lower molecular weight substances (potential humics) were completed prior to protein evaluation. Spectroscopic scans were then performed on filtered teas through the UV and higher visible range (290-500 nm) to determine the presence of oxygen-binding proteins.
Results/Conclusions
Positive tests of the Pierce bicinchoninic acid (BCA) protein assay using the unfiltered teas suggested the presence of protein in L. maackii teas, but not in teas of native plant leaves. A BCA protein assay was also run on the filtered teas, but had a negative result for the presence of protein in both the Amur Honeysuckle and native tree teas. SDS-PAGE gel electrophoresis silver assays was used to validate protein presence and to determine the molecular size of the compounds within the leaves. However, the gels using both the filtered and unfiltered teas showed no presence of proteins within either the Amur Honeysuckle tea or the native trees tea. These tests have helped us conclude that the oxygen-binding particle is not a protein. Further testing will be needed in order to determine what type of particulate released during the decomposition of L. maackii is binding oxygen.