Dextran-bound adenosine, inosine, and nebularine have been prepared by carbodiimide coupling of their 2',3'-O-(4-carboxyethyl-l-methylbutylidene) cyclic acetal derivatives to 6-aminohexyldextran or 12-aminododecanyldextran. The latter polymers were prepared by cyanogen-bromide activation of dextran T80 followed by reaction with 1,6-diaminohexane or 1 ,12-diaminododecane. A high CNBr concentration leads to high-molecular-weight material, probably due to cross-linking, accompanied by a decrease in the digestion velocity using endo-dextranase from Penicillium species (EC 3.2.1.1 1).The dextran-bound nucleosides, as well as the nucleoside 2', 3'-O-(4-ethoxycarbonyI-l -methylbutylidene) acetal derivatives, were tested as substrates and inhibitors for adenosine deaminase. The K, of the adenosine acetal ester is identical to that of adenosine which shows that acetalation does not hinder complex formation. Since the maximum velocity of deamination is decreased fourfold, the modified substrate does not fit as well as the nucleoside. The polymer-bound acetals show a 3 -8-fold increase of K, or Ki and unchanged V compared to the corresponding acetals while dextranase digestion of the support does not alter the kinetic data. This indicates that the length of the polysaccharide chain does not interfere either with the complex formation or with the catalytic activity of the modified substrate. Since the activation energies of the deamination reactions of adenosine, its acetal ester, and dextran-linked adenosine are all similar (29.8 -32.3 kJ mol-') it is concluded that no diffusion control of the enzymatic reaction results from the binding of the nucleoside acetals to dextran T80.Heterogeneous enzyme-catalysed reactions can be divided into two categories: (a) reactions between soluble substrates and enzymes embedded in biological membranes or other native complex structures ; (b) reactions between a soluble enzyme and a polymer-bound or insoluble substrate [l -61.This prompted us to immobilise substrates and inhibitors of adenosine deaminase via cyclic acetal derivatives onto soluble dextran T80. Dextrans are a group of structurally related polysaccharides (mr, lo5 -10') elaborated by various microorganisms, especially by strains of Leuconostoc. All dextrans contain linear chains of 1 .+ 6-linked a-D-glucopyranose residues as the dominant structural feature [7]. Since we have already immobilised adenosine deaminase on dextran T80 [8], we now have a complete kinetic model system in which either the enzyme or its binding partners are covalently bound to a polymer matrix.The polymer attachment of nucleosides and nucleoside antimetabolites has several implications. (a) From the pharmacological point of view, the brief plasma persistance of small substrates and inhibitors can be overcome by linking the biomolecule to a polymer such as dextran T80, which is used as a blood volume expander. High activity of the modified substrate can only be expected if the position of attachment is carefully chosen and, if possible, a stereochemica...

Dextraii-linked adenosine deaminase (EC 3.5.4.4) has been prepared. The polymer-linked enzyme possesses an optiiiial cnzymatic activity of 27 units/mg immobilized protein (noii-bound enzyme : 200 units/mg protein). Support-bound adenosine deaminase (4.5 pg protein/mg dextran) shows an enhanced heat stability, a moderately -increased h',,,? and a decreased Vvalue compared to those of the free enzyme. The pH dependences of V and pK, values of dextran-linked adenosine deaminase show only two inflection points compared to three for the free enzyme, which are equivalent to the pK values of the enzyme. Since the missing third inflection point (pH 9.8) can be assigned to the pK value of the t:-amino group of lysyl residues, it can be concluded, that immobilization of adenosine deaminase on cyanogen-bromide-activated dextran took place via these lysyl residues. The remaining pK values found from tlie other inflection points are moderately shifted owing to the altered secondary structure. From the temperature dependence of the enzymatic activity, a 40% decrease of the activation energy of the support-bound enzyme was found, indicating diffusion controlled deamination. The immobilization of adenosine deaminase results in a fluorescence quenching of 80 x,, without shifting the ultraviolet maximum of the emission spectrum. As already shown for unmodified dextran, the matrix of polymer-linked adenosinc deamiiiase is degradable by a bacterial endodextranase (EC 3.2.1.1 1).Applications of immobilized enzymes for medical u;e have been exten3ively .itudied and developed during the last couple of year., [1,2], since the attachment of protein3 to carbohydrates increase> their circulatory lifetimes and reduces tlie distribution of the enzyme over the compartments of the organism [3,4].Besides thib, biologically active proteins artificially bound to high-molecular-weight carriers are of theoretical interest, .ince thcy may scrvc a s model sydenis for proteins embedded in biological membranes or other native complex structures like glycoproteins [3,5]. Adenosine deaminase, an enzyme with a molecular weight of 35000, catalyses nucleophilic displacement of tlie 6-amino group of adenosine by water, yielding inosine, within a pH range of 4.5-9.6 via a tetrahedral transition state. At pH 9 tlie enzyme even catalyze\ the reverse reaction in tlic presence of ammonia [G-81. By immobilization of adenosine deaminaae to soluble polymer3. the p l a m a half-lif'es (rliz) should be increased. Tliercfore we coupled adenosine deaminase to a soluble dextran. Dextrans are a group of structurally related polysaccharides ( M ,