Front Matter

affect the activity of either form of the enzyme in case of low diheptanoyl-PE con-

centrations. The data obtained with diheptanoyl-PC suggest that the behavior of

immobilized and soluble PLA 2 against monomeric phospholipids is similar; how-

ever, the activity on micellar substrate differs by more than one order of magni-

tude. This tremendous increase in PLA 2 activity towards substrate molecules orga-

nized into lipid – water interfaces was shown previously by Verhey et al. (1981). An

analysis of the kinetic data for substrate concentration above the CMC-value re-

vealed that the apparentKmvalues for native and immobilized PLA 2 were simi-

lar, whereasVmaxfor the soluble PLA 2 was about 10-fold higher.

The poor residual activities found for immobilized PLA 2 (between 1 and 2 %)

raises the question of whether this is due to a mass transfer resistance produced

by the carrier matrix, to partitioning effects, or to the applied coupling method itself

(see Section 13.3.3). Ferreira et al. (1993) were the first to repeat the experiments of

Lombardo and Dennis. However, in contrast to the latter, they covalently bound a

cobra venom enzymeNaja naja kaouthiaPLA 2 toN-hydroxysuccinimide-activated

agarose (Biorad) with enzyme fixation primarily throughe-amino groups and to

diaminodipropylamine-derivatized agarose (Pierce) by use of a carbodiimide where

enzyme binding occurs through carboxylic residues. The results of Lombardo and

Dennis could be confirmed in so far as the binding of PLA 2 via succinimide was

accompanied by a strong activity loss. Both succinimide and tresyl chloride use

thee-amino groups of lysine residues to attach the enzyme to the carrier surface.

By contrast, with the carbodiimide method PLA 2 -preparations with an activity re-

tention of 50 % were obtained, clearly indicating that the chemistry of enzyme bind-

ing is crucial in this case. A pH shift of the microenvironment could be excluded as

the activity – pH profiles for both types of PLA 2 were nearly identical. A very im-

portant finding was that the carbodiimide-bound PLA 2 exhibited interfacial recogni-

tion in the presence of short-chain phospholipid micelles, as is characteristic for the

soluble form. From this it can be deduced further that a substrate-induced aggrega-

tion of PLA 2 molecules to dimers – or higher-order aggregates – obviously is not

linked to the interfacial activation observed at concentrations around the CMC-

value. A corresponding hypothesis discussed (controversially) for a long time

was formed on the basis of chemical modification of PLA 2 (Roberts et al., 1977)

and other experiments (van den Bosch, 1982). These results also showed that

such experiments may contribute to the enlightenment of theoretical questions

such as the mechanism of enzymatic catalysis.

It can be demonstrated simply by adsorption experiments that the activity of cova-

lently bound PLA 2 is mainly effected by the type of chemistry used. Adsorption of

PLA 2 (porcine pancreas; Lecitase, from Novo) to a highly dispersive silicon oxide

(Cab-osil M-5, Fluka) with a surface size of 200 m^2 g–1yielded catalytically active

products (see Figure 2). The residual activities of PLA 2 adsorbed from an aqueous or

from a Tris/HCl buffer solution (pH 9) were 15 % and 26 %, respectively. Lowering

the polarity of the reaction milieu by addition of water-miscible organic solvents

such as ethanol led to an increase in the activity of adsorbed PLA 2 to above

50 % (unpublished results). However, these preparations were not suited for contin-

uous use because of the weak binding forces between carrier surface and enzyme.

The fact that the temperature behavior of adsorbed PLA 2 is the same as found for the

soluble enzyme with respect to the activation energy and the temperature optimum is

13.4 Immobilization of phospholipases 275