Sequential Conjugation of Two Different Types of Catalytically Active Enzymes to Liposomes via Bis-Aryl Hydrazone Bonds.

Controlled chemical conjugation of enzymes to the external surface of liposomes as simple biomembrane model systems and the investigation of the catalytic activity of such liposome-bound enzymes in vitro is one of the approaches toward a better understanding of enzymatic transformations occurring at biointerfaces in vivo. Liposomes with surface-bound enzymes may also find applications as soft, biodegradable immobilized enzyme systems for reactions carried out under mild conditions in an aqueous medium. In the present work, phosphoenolpyruvate carboxylase from microorganisms (PEPC) modified with 6-hydrazinonicotinate acetone hydrazone (PEPC-HyNic) was conjugated at 25 °C and pH = 7 to liposomes of about 100 nm diameter modified with 4-formylbenzoate (liposome-4FB) via the bis-aryl hydrazone (BAH) bond that forms upon reaction of HyNic with 4FB. The BAH bond formation and colloidal stability were followed for a dispersion of liposome-4FB reacting with PEPC-HyNic through monitoring of the time-dependent changes of the UV/vis absorption spectrum, yielding catalytically active liposome-BAH-PEPC after chromatographic purification. Using the previously elaborated conditions for the preparation of (i) HyNic-modified bovine carbonic anhydrase (BCA-HyNic) and (ii) liposomes with surface-bound BCA (liposome-BAH-BCA), in the present work, liposomes containing both enzymes, BAH-bonded PEPC as well as BAH-bonded BCA, were prepared by sequentially attaching the two enzymes to the liposomes. For this, two approaches were applied. In the first approach, PEPC was first attached to the liposomes, followed by the attachment of BCA, yielding liposome-BAH-PEPC1·BCA2. In the second approach, BCA was first attached, followed by PEPC, yielding liposome-BAH-BCA1·PEPC2. For comparison, both enzymes were also attached simultaneously, yielding liposome-BAH-(PEPC·BCA). All three types of liposomes were characterized in terms of activity and stability of the two enzymes. The most successful approach turned out to be the one in which PEPC was attached first to the liposomes, liposome-BAH-PEPC1·BCA2. For the elaborated experimental conditions, each outer liposome surface contained on average about 2-3 active PEPC and about 100 active BCA molecules.