HR: 1330h
AN: NB33L-14    [Abstracts]
TI: Comparison of Alkalizing Solutions Used in Extracellular Enzyme Assays
AU: * Johnson, A C
EM: ajohns24@emich.edu
AF: Department of Biology, Eastern Michigan University, 316 Mark-Jefferson, Ypsilanti, MI 48197 United States
AU: Kuehn, K A
EM: kkuehn@emich.edu
AF: Department of Biology, Eastern Michigan University, 316 Mark-Jefferson, Ypsilanti, MI 48197 United States
AU: Francoeur, S N
EM: steve.francoeur@emich.edu
AF: Department of Biology, Eastern Michigan University, 316 Mark-Jefferson, Ypsilanti, MI 48197 United States
AB: Extracellular enzymatic activity plays an important role in microbial nutrient cycling, and is commonly studied through the use of fluorogenic substrates. Samples are generally alkalized to maximize fluorescence yield and to prevent among sample variability in pH from affecting sample fluorescence. It is also frequently assumed that the alkaline solutions stop enzymatic reactions. Different alkalizing agents (pH 10 sodium carbonate-bicarbonate buffer, 0.05M glycine-ammonium hydroxide buffer, 40mM sodium hydroxide solution, and 2M sodium hydroxide solution) were explored for their ability to halt periphytic enzymatic activity without degrading the fluorescent product. L-Leucine-7-amido-4-methylcoumarin hydrochloride (LAMC) and 4-Methylumbeliferyl phosphate disodium salt (MUF-P) were used as fluorogenic substrates. Although 2M NaOH solution appeared to stop enzymatic reactions, it completely degraded both the AMC and MUF flurochromes, rendering it unsuitable for use as an alkalizing agent. The remaining three buffers did not greatly erode either fluorochrome, however, they were unable to halt enzymatic activity reliably. This brings into question the method of using alkalizing agents as a means of halting enzymatic reactions for later analysis.
DE: 4239 Limnology
DE: 4840 Microbiology
DE: 4894 Instruments and techniques
DE: 9810 New fields (not classifiable under other headings)
SC: North American Benthological Society [NB]
MN: 2005 Joint Assembly