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Reconstitution in the apo-enzymes was performed overnight at four °C prior to elimination of unbound metal by passage by way of a PD-10 column. The PD-10 column was pre-handled with dipicolinate to eliminate traces of unbound metal and then washed with five column volumes of metallic cost-free HEPES buffer, pH eight.0. Soon after elution, the concentration of the steel-reconstituted enzyme was resolute by UV absorbance as well as metal content on the samples determined by ICP-MS.
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-trifluoroacetyl substituted substrate is hydrolyzed 26 moments quicker compared to organic substrate although the N
m values have been decided from matches of the info to the straight line. Saturation curves showing substrate inhibition had been suit to equation two had been K
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