Prosthetic group determination

HRP is a hemoprotein containing photohemin IX as its prosthetic group. The presence of the heme structure gives the enzyme its characteristic color and maximal absorptivity at 403 nm.The ratio of its absorbance in solution at 403 nm to its absorbance at 275 nm, called the RZ or Reinheitzahl ratio, can be used to approximate the purity of the enzyme. However, at least seven isoenzymes exist for HRP (Shannon et al., 1966 Kay et al., 1967 Strickland et al., 1968), and their RZ values vary from 2.50 to 4.19. Thus, unless the RZ ratio is precisely known or determined for the particular isoenzyme of HRP utilized in the preparation of an antibody-enzyme conjugate, subsequent measurement after crosslinking would yield questionable results in the determination of the amount of HRP present in the conjugate. 

A second example of a membrane-bound arsenate reductase was isolated from Sulfurospirillum barnesii and was determined to be a aiPiyi-heterotrimic enzyme complex (Newman et al. 1998). The enzyme has a composite molecular mass of 100kDa, and a-, P-, and y-subunits have masses of 65, 31, and 22, respectively. This enzyme couples the reduction of As(V) to As(III) by oxidation of methyl viologen, with an apparent Kra of 0.2 mM. Preliminary compositional analysis suggests that iron-sulfur and molybdenum prosthetic groups are present. Associated with the membrane of S. barnesii is a h-type cytochrome, and the arsenate reductase is proposed to be linked to the electron-transport system of the plasma membrane. 

Because xenobiotic metabolism involves many enzymes with different cofactor requirements, prosthetic groups, or endogenous cosubstrates, it is apparent that many different nutrients are involved in their function and maintenance. Determination of the effects of deficiencies, however, is more complex because reductions in activity of any particular enzyme will be effective only if it affects a change in a rate-limiting step in a process. In the case of multiple deficiencies, the nature of the rate-limiting step may change with time... 

Shown in Figures 5-7 are the redox pathways for xanthine oxidase, sulfite oxidase, and nitrate reductase (assimilatory and respiratory), respectively. These schemes address the electron and proton (hydron) flows. The action of the molyb-doenzymes is conceptually similar to that of electrochemical cells in which half reactions occur at different electrodes. In the enzymes, the half reactions occur at different prosthetic groups and intraprotein (internal) electron transfer allows the reactions to be coupled (i.e., the circuit to be completed). In essence, this is the modus operandi of these enzymes, which must be determined before intimate mechanistic considerations are seriously addressed. 

Some enzymes require cofactors for the activities Enzymes that require covalent cofactors (prosthetic groups, e.g., heme in cytochromes) or non-covalent cofactors (coenzymes, e.g., NAD(P)+ in dehydrogenases) for activities are called haloenzymes (or simply enzymes). The protein molecule of a haloenzyme is termed proenzyme. The prosthetic group/coenzyme dictates the reaction type catalyzed by the enzyme, and the proenzyme determines the substrate specificity. 

One of the cellobiose oxidoreductases present in S. pulverulentum has been characterised and named cellobiose oxidase (Ander and Eriksson, 1978). The enzyme contains both haem and flavin co factors and binds irreversibly to concanavalin A-Sepharose, suggesting that it is a glycoprotein. Cellobiose oxidase from S. pulverulentum has now been purified to homogeneity by Morpeth (1985). The carbohydrate and amino acid compositions of the enzyme have been determined. The enzyme contains FAD and cytochrome b prosthetic groups and is a monomer with an Mr of 74400 determined by sedimentation equilibrium. 

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