A Fluorometric Assay to Compare SUMO Isoform Preferences in the SENP/ULP Family

Comparison of proteolytic activities of deconjugating enzymes toward ubiquitin or other ubiquitin-like modifiers (UbL), such as SUMO isoforms or Nedd8, has been a central focus in recent years to understand the dual or even multiple activities exhibited by members of the SENP/ULP family. This protocol outlines a competitive assay for an initial kinetic assessment of a SENP/ULP family member with specificity for alternative SUMO substrates. By using this method, the affinity of a SENP/ULP member for different SUMO isoforms or SUMO mutant variants can be compared. Also, IC₅₀ values from competition experiments can be extracted to gain initial insights into the SENP/ULP substrate specificity. This is particularly useful when the K_M (Michaelis constant) of the reaction is high and it is difficult to achieve high substrate concentrations. This protocol can also be applied to other CE-clan proteases with dual or multiple activities, such as those found in bacteria, to compare preferences between different types of UbL modifiers like ubiquitin, SUMO, or Nedd8.