Stabilization of creatine kinase encapsulated in silicate sol-gel materials and unusual temperature effects on its activity

The enzyme creatine kinase (CK) is stabilized by encapsulation in silicate sol-gel monoliths. Its activity is measured as a function of long-term storage time at both room temperature and at elevated temperatures and is compared to that in solution. At room temperature, the activity of the encapsulated enzyme decreases to 50% of its initial value after more than 5 months, whereas in solution it decreases to 50% after 10 days. At 47 degreesC, the immobilized enzyme retains 50% of its maximum activity after 5 days of constant heating compared to that at 13 h in solution. At 60 T, the immobilized enzyme retains 50% of its maximum activity after 5 h of heating as compared to that for less than an hour in solution. Surprisingly, a 4-fold increase in activity is observed after short exposures to the elevated temperatures. This increase is explained by structural changes in both the enzyme and the sol-gel matrix. The structural integrity and conformational changes of the encapsulated enzyme are observed by circular dichroism spectroscopy. The spectrum shows that the initially encapsulated enzyme has a structure different from that in solution but that upon heating the enzyme reverts to a conformation similar to that in solution. In addition, the encapsulated enzyme does not completely denature at temperatures up to 90 degreesC while in solution the midpoint temperature of the unfolding transition is 75 degreesC. These effects are interpreted in terms of electrostatic interactions between the positively charged patches on the enzyme's surface and the sol-gel matrix and to conformational changes within the pores upon heating. Heat treatment also affects the silica matrix by increasing the pore size as measured by gas absorption/desorption isotherms. The increase may allow small changes in the enzyme's structure, but in general the pore constrains the enzyme and inhibits denaturation.