Kinetics of enzyme attack on substrates covalently attached to solid surfaces: influence of spacer chain length, immobilized substrate surface concentration and surface charge.

The use of α-chymotrypsin to cleave covalently bound N-acetyl-l-tryptophan (Ac-Trp-OH) from the surfaces of aminopropylated controlled pore glass (CPG) and the polymer PEGA1900 was investigated. Oligoglycine spacer chains were used to present the covalently attached Ac-Trp-OH substrate to the aqueous enzyme. In the absence of the oligoglycine spacer chain, the rate of release was relatively slow, especially from the PEGA1900. These slow rates reflect the position of the amino group to which Ac-Trp-OH is covalently attached. On the glass there was a clear optimum with a chain of four glycine residues. For PEGA1900 there is no real apparent change beyond two glycine residues. The decline in rate beyond these optima are a possible result of changes in oligoglycine structure. Comparing different surface loadings of bound substrate the rate of release of Ac-Trp-OH from CPG with a pore diameter of 1200 A was optimal when using 83% of the maximum that can be coupled, then fell again at higher loading. The rate o...