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Free keywords:
Proteases; Quantumdot; FRET; Enzyme; Sensor; Michaelis-Menten
Abstract:
As an enzyme superfamily, proteases are rivaled only by kinases in terms of their abundance within the human genome. Two ratiometric quantum dot (QD) Forster resonance energy transfer-based sensors designed to monitor the activity of the proteolytic enzymes collagenase and elastase are investigated here. Given the unique material constraints of these sensing constructs, assays are realized utilizing excess enzyme and fixed substrate in progress curve format to yield enzyme specificity or k (cat)/K (m) ratios. The range of k (cat)/K-m values derived is 0.5-1.1 mM(-1) s(-1) for the collagenase sensor and 3.7-4.2 mM(-1) s(-1) for the elastase sensor. Of greater interest is the observation that the elastase sensor can be well represented by the Michaelis-Menten model while the collagenase sensor cannot. The latter demonstrates increased specificity at higher peptide substrate/QD loading values and an apparent QD-caused reversible inhibition as the reaction progresses. Understanding the detailed kinetic mechanisms that underpin these types of sensors will be important especially for their further quantitative utilization.
