By examining the properties of the proteins packaged inside of the vault particle, it could be demonstrated that these proteins are functionally sequestered into a protective environment. This has been shown in two ways: one enzymatic and the other spectroscopic. The enzymatic properties of LUC-INT were examined under native conditions and following packaging into the vault lumen (referred to here as LUC-INT vaults). The figure (at right) summarizes a group of kinetic experiments using the two substrates of the luciferase reaction luciferin and ATP. Addition of these substrates to free LUC-INT and to LUC-INT vaults resulted in formation of the expected chemilumenescent product, oxyluciferin (A). However, the kinetics of oxyluciferin production was different in the two enzyme preparations. Free LUC-INT catalyzed a rapid formation of product that slowly decayed over time (B, blue line), while LUC-INT vaults produced product much more slowly, peaking only after several minutes (B, red line). These kinetics suggested that accessibility to LUC-INT inside the vault might be limited to one or both of the substrates. To examine this a series of pre-incubation experiments were carried out. Preincubation of LUC-INT vaults with ATP, prior to addition of luciferin, resulted in product formation much like that seen with free LUC-INT, indicating that accessibility of ATP to the vault lumen was limited. Apparently the vault acts as a partial diffusion barrier to this charged molecule.
Similar findings were seen when vaults packaged with the GL-INT protein were examined spectroscopically. A Stern-Volmer kinetic analysis of the fluorescence quenching of free GL-INT and GL-INT vaults (not shown) indicated a very large quenching constant for GL-INT vaults indicative of "super quenching", suggesting that multiple copies of GL-INT protein are brought together in close proximity in the central lumen of the vault particle. The rate of quenching of free GL-INT and GL-INT vaults was strikingly different. This is shown in the figure at right (bottom panel). Here the quenching by KCl, a contact-dependent quencher, of packaged GL-INT required hundreds of seconds (red line), compared to the nearly instantaneous quenching of free GL-INT (blue line). Here too, the data indicate that the lumen of the vault is a protective environment that serves as a partial diffusion barrier to charged molecules (Kickhoefer et al., 2005).