Amidinohydrolases

In an early application, an enzyme electrode system was reported for the determination of creatinine and creatine, using a combination of creatinine amidohy-drolase, creatine amidinohydrolase and sarcosine oxidase, co-immobilized on an asymmetric cellulose acetate membrane. Thus, the hydrogen peroxide produced was detected to give a quantitative measure of creatine and creatinine in biological fluids [70]. 

The official EC name of this enzyme is L-arginine amidinohydrolase, the last word refers to the fact that the amidino group (dotted circle in the equation) is cleaved from arginine by introduction of a molecule of water across the C—N bond. In the reaction, a nonpeptide C—N bond is cleaved thus, the second EC number for arginase is 5 its whole classification number is 3.5.3.1. 

Poly(2,5-dim ethoxy aniline] Creatinine amidohydrolase Overoxidized PPy Creatine amidinohydrolase... 

Sarcosine is also formed in the hydrolysis of creatine mediated by creatine amidinohydrolase (creati-nase). This reaction is used in a variety of sensors for creatine and creatinine. 

Creatinine amidohydrolase hydrolyzes creatinine to creatine, the creatine is hydrolyzed to sarcosine, urea by creatine amidinohydrolase, and sarcosine oxidase catalyzes the breakdown of the sarcosine to glycine, formaldehyde, and peroxide. When horseradish peroxidase is included, the reduction of peroxide can be coupled with various compounds to produce absorbing species. One example of a suitable chromogen is 3,5-dichloro-2-hydroxy-benzenesulfonic acid-4-aminophenazone, with the absorbance being measured at 510 nm. 

The preferred absorption-based methods are those based on enzyme-catalyzed reactions. The absorbance of NADH (340 nm) after addition of creatine amidinohydrolase, sarcosine oxidase, formaldehyde dehydrogenase, and NAD to a creatine-containing solution is proportional to the concentration of creatine present. Creatinine amidohydrolase produces creatine from creatinine. Thus, any assays for creatinine which use creatinine amidohydrolase in the first step can be used for creatine by just leaving out that first step. Conversely, creatine can interfere in those analyses, although creatine is normally present in substantially lower concentrations than creatinine. 

When the three-enzyme sequence based on creatinine amidohydrolase is used, any creatine present can interfere with the determination of creatinine, so two sensors are used one to determine the total creatine plus creatinine and one to determine just creatine (by only using creatine amidinohydrolase and sarcosine oxidase). Creatinine is determined by difference. Amperometric sensors are generally based on this sequence and do not suffer from interferences. They are usually designed to respond to peroxide, though some have used oxygen electrodes. Typically, Pt electrodes are used. A sensor for just creatine only requires the creatine amidinohydrolase and sarcosine oxidase sequence. 

Creatinine amidohydrolase Creatine amidinohydrolase Sarcosine oxidase Horseradish peroxidase 3,5-Dichloro-2- hydroxybenzenesulfonic acid/4-aminophenazone... 

Creatinine amidohydrolase Creatine amidinohydrolase Creatine amidinohydrolase Creatine kinase Sarcosine oxidase Sarcosine oxidase Pyruvate kinase Horseradish peroxidase Horseradish peroxidase Lactate dehydrogenase NADH Methylene blue Benzoquinone-imine... 

A three-enzyme electrode system, such as needed for creatinine measurement, poses a more difficult enzyme-immobilisation problem, in that different enzymes have different immobilisation requirements and their microenvironmental interrelationships need to be optimised. For one creatine sensor, the requisite creatine amidinohydrolase and sarcosine oxidase were immobihsed in polyurethane pre-polymer and PEG-hnked creatinine amidohydrolase was attached via diisocyanate pre-polymer to create a polyurethane adduct [14]. The likelihood of enzyme inactivation with chemical immobih-sation is high, but provided an enzyme preparation survives this, long-term stability is feasible. In the case of these three particular enzymes, a loss of activity resulted from silver ions diffusing from the reference electrode the material solution was to protect the enzyme layer with a diffusion-resisting cellulose acetate membrane. 

Figure 3.1 Interference removing oxidising membrane based on incorporated Pb02 particles over a three-enzyme creatinine biosensor based on creatinine amidohydrolase (CA), creatine amidinohydrolase (Cl) and sarcosine oxidase (SA).

The coreticulation described above [3] is simple to perform during multienzymatic immobilization. The required enzymes are mixed with albumin, and enough glutaraldehyde is added to cross-link the various proteins. This method is also used for the coimmobilization of creatinin amidohydrolase, creatin amidinohydrolase and sarcosine oxidase, to construct a creatinin electrode, and for the coimmobilization of a-glucosidase and glucose oxidase for the determination of starch [40]. 

---