With the key role that DBH plays in the biosynthesis

With the key role that DBH plays in the biosynthesis of catecholamines, this process has been subject of a number of studies. The majority of the assays utilize crude extracts giving rise to the possibility of endogenous inhibitors in the extracts that can interfere with DBH activity [6]. When highly purified enzyme is used it involves long and complicated purification procedures which are costly. The enzymatic assays can also be cumbersome, for example, one assay utilizes a two-step enzyme method, using phenylethanolamine N-methyltransferase and S-adenosyl-l-methionine [6]. However, with these techniques saturating substrate concentrations for DBH cannot be examined due to the inhibition of the second enzyme, phenylethanolamine N-methyltransferase. The main problem faced by researchers when characterizing DBH is the repeated need for large amounts of highly purified enzyme. However, there are recent applications that utilize SB 203580 in one or more immobilized form [7], [8]. The enzymes were immobilized onto solid supports and used in batch incubations [7], [9] or as biosensors (cf. Refs. [8], [9], [10], [11], [12], [13]). These applications do not require highly purified enzymes and decrease the amounts of enzyme required. The immobilized enzymes retain their activity and can be reused following a simple washing procedure. In this study, the hydrophobic character of the IAM stationary phase was used to immobilize commercially available partially purified dopamine β-hydroxylase. The IAM interphase is derived from the covalent immobilization of 1-myristoyl-2-[(13-carboxyl) tridecanoyl]-sn-3-glycerophospholine on aminopropyl silica, and resembles one-half of a cellular membrane [14]. The hydrocarbon chains create interstitial spaces that allow for the insertion of DBH. DBH was also immobilized onto glutaraldehyde-P (Glut-P), a wide-pore silica that has been covalently clad with polyethyleneimine, a hydrophilic polymer [15]. The reactive amine groups of the polymer form a covalent bond with glutaraldehyde. This particular support is ideal for immobilization of proteins with primary amino groups that form an amine–aldehyde Schiff linkage with Glut-P [15].
Materials and methods
Results
Discussion Many assays in the literature for DBH require the use of catalase to protect the active site from hydrogen peroxide, which is a by-product in the initial step of the reaction. For the non-immobilized enzyme there was a visible increase in the rate with increasing amounts of catalase. However, for both types of immobilized enzyme minimal amounts of catalase are required for maximal activity. The rates for both forms of immobilized enzyme (see Table 1) are lower relative to the non-immobilized DBH. This suggests the enzyme is working slower and therefore the amount of hydrogen peroxide produced as a by-product is minimal and does not affect the enzyme activity. The immobilized enzymes have conformations that do not require large amounts of catalase for protection.