The shortening of the 3'-end poly(A) tail, also called deadenylation, is crucial 
to the regulation of mRNA processing, transportation, translation and 
degradation. The deadenylation process is achieved by deadenylases, which 
specifically catalyze the removal of the poly(A) tail at the 3'-end of 
eukaryotic mRNAs and release 5'-AMP as the product. To achieve their 
physiological functions, all deadenylases have numerous binding partners that 
may regulate their catalytic properties or recruit them into various protein 
complexes. To study the effects of various partners, it is important to develop 
new deadenylase assay that can be applied either in vivo or in vitro. In this 
research, we developed the deadenylase assay by the size-exclusion 
chromatography (SEC) method. The SEC analysis indicated that the poly(A) or 
oligo(A) substrate and the product AMP could be successfully separated and 
quantified. The enzymatic parameters of deadenylase could be obtained by 
quantifying the AMP generation. When using the commercial poly(A) as the 
substrate, a biphasic catalytic process was observed, which might correlate to 
the two distinct states of poly(A) in the commercial samples. Different lots of 
commercial poly(A) had dissimilar size distributions and were dissimilar in 
response to the degradation of deadenylase. The deadenylation pattern, 
processive or distributive, could also be investigated using the SEC assay by 
monitoring the status of the substrate and the generation kinetics of AMP and 
A2. The SEC assay was applicable to both simple samples using the purified 
enzyme and complex enzyme reaction conditions such as using protein mixtures or 
crude cell extracts as samples. The influence of solutes with absorption at 254 
nm could be successfully eliminated by constructing the different SEC profiles.