A Comparison of Polymer Blocking Agents in the in vitro Motility Assay

Abstract

Blocking agents are used in in vitro motility assays to stabilize the motor proteins myosin or heavy meromyosin (HMM) and to prevent non-specific binding of actin to regions of microscope coverslip that are devoid of motors. Bovine serum albumin (BSA) or casein is typically used for blocking, but there is occasional need for non-protein blocking agents. We compared skeletal myosin and HMM function in motility assays as a function of the blocking agent that was used; these blocking agents included polysorbate (Tween) 20 and six different molecular weights of polyvinyl pyrrolidone (PVP) ranging from 10 kDa to 1.3 MDa, as well as BSA and β-casein as controls. In vitro motility assays were preformed and actin filament movement was quantified using automated particle tracking algorithms. PVPs of all molecular weights supported the motility of both HMM and myosin, though there was a slight downward trend in mobility at the highest molecular weights. When HMM was used in the motility assay, Tween showed poor mobility (1.7 um/s) compared to BSA (9.4 um/s). In contrast, full length myosin showed high mobility when blocked with Tween (8.3 um/s) compared to BSA (6.6 um/s). To determine whether Tween is a direct inhibitor of HMM function, NH4-activated ATPase assays were performed in solution with either BSA or Tween. There was no significant difference in ATPase rates between these two conditions. However, when the NH4-activated ATPase assay was repeated with HMM bound to a flow cell, Tween inhibited the ATPase activity. Thus while Tween does not directly inhibit the motor domains of HMM, it may adversely alter its binding to hydrophobic surfaces. In conclusion, low- to mid-molecular weight PVPs are excellent polymer blocking agents for actin-myosin or actin-HMM motility assays.