Abstract
Protease levels in human blood are often prognostic indicators of inflammatory, thrombotic or oncogenic disorders. The measurement of such enzyme activities in substrate-based assays is complicated due to the low prevalence of these enzymes and steric hindrance of the substrates by the more abundant blood proteins. To address these limitations, we developed a molecular construct that is suitable for microsphere-cytometer based assays in the milieu of human blood plasma. In this proof of principle study, we demonstrate the utility of this substrate to measure metalloprotease ADAMTS13 activity. The substrate, expressed in E. coli as a fusion protein, contains the partial A2-domain of von Willebrand factor (VWF amino acids 1594–1670) that is mutated to include a single primary amine at the N-terminus and free cysteines at the C-terminus. N-terminus fluorescence conjugation was possible using NHS (N-hydroxysuccinimide) chemistry. Maleimide-PEG(Polyethylene glycol)n-biotin coupling at the C-terminus allowed biotinylation with variable PEG spacer lengths. Once bound to streptavidin-bearing microspheres, the substrate fluorescence signal decreased in proportion with ADAMTS13 concentration. Whereas recombinant ADAMTS13 activity could be quantified using substrates with all PEG repeat-lengths, only the construct with the longer 77 PEG-unit could quantify proteolysis in blood plasma. Using this longer substrate, plasma ADAMTS13 down to 5% of normal levels could be detected within 30 min. Such measurements could also be readily performed under conditions resembling hyperbilirubinemia. Enzyme catalytic activity was tuned by varying buffer calcium, with lower divalent ion concentrations enhancing cleavage. Overall, the study highlights the substrate design features important for the creation of efficient proteolysis assays in the setting of human plasma. In particular, it emphasizes the need to introduce PEG spacers in plasma-based experiments, a design attribute commonly ignored in immobilized peptide-substrate assays.
Highlights
The total protein concentration of human blood plasma is 60–80mg/mL with serum albumin, globulins, transferrin, fibrinogen and a handful of additional molecules constituting ~99% of the total content [1,2]
ADAMTS13 is a constitutively active blood metalloprotease that cleaves ultralarge von Willebrand factor (VWF) in circulation to produce VWF units with smaller molecular mass [3,4]. The absence of this protease prevents the breakdown of VWF and this contributes to life-threatening thrombosis in microvessels, a disorder called thrombotic thrombocytopenic purpura (TTP)
This study describes a novel E. coli derived substrate that can be applied in microsphere-cytometry assays to detect protease activity in human blood plasma
Summary
The total protein concentration of human blood plasma is 60–80mg/mL with serum albumin, globulins, transferrin, fibrinogen and a handful of additional molecules constituting ~99% of the total content [1,2]. A much smaller portion of this complex mixture (
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