Ceruloplasmin enzymatic determination

The results of enzymatic determinations of ceruloplasmin are often expressed in arbitrary units, and the values judged in the light of a series of results obtained in normal subjects by the same method. Expression of the enzyme activity in milligrams of ceruloplasmin per unit volume of serum is also possible. The relation between oxidase activity and the amount of ceruloplasmin in serum can be determined by measuring in parallel samples of sera both the oxidase activity and the change of optical density at 610 mix before and after the addition of ascorbic acid or cyanide. On the basis of the known absorbancy index, the ceruloplasmin concentration can be calculated (see Section 2.2.1) and the relation between it and the enzyme activity determined. Alternatively, purified human ceruloplasmin can be used for standardization of the enzymatic method. The ceruloplasmin content of the purified preparation can be determined colorimetrically or, in the case of a highly purified preparation, by nitrogen analysis. Predetermined increments of ceruloplasmin can then be added to aliquots of a selected serum. It is convenient to select a serum with relatively low ceruloplasmin level to start with. Serum of a patient with Wilson s disease, some of whom have no measurable amount of enzyme activity, would be ideal for the purpose however, Walshe (W5) has recently found an inhibitor in these sera. 

The estimation of the empirical parameters is a general problem of least squares estimation by linear models. The basics are introduced in Section 6.2. With the parameters in hand, the model can be used to plot the response in dependence on the individual factors. The RSM is explained here by further exploring the enzymatic determination of ceruloplasmin from the previous section about screening designs. 

Figure 4.19 Simplex search for optimum PPD concentration and pH value for the enzymatic determination of ceruloplasmin (cf. Example 4.7).

Solberg and co-workers have applied discriminate analysis of clinical laboratory tests combined with careful clinical and anatomic diagnoses of liver disease in order to determine which combinations of the many dozen liver diagnostic tests available are the bes t ( ). These authors found that the measurement of GPT, GMT, GOT, ALP and ceruloplasmin were the most useful enzymatic tests, when combined with other non-enzymatic tests such as the measurement of bilirubin, cholesterol, hepatitis-B associated Australian antigen, etc. Another group of highly useful enzymes, not discussed in this review, are those clotting factors and the enzyme cholinesterase which are synthesized by the liver cells. 

Various spectroscopic methods have been used to probe the nature of the copper centers in the members of the blue copper oxidase family of proteins (e.g. see ref. 13). Prior to the X-ray determination of the structure of ascorbate oxidase in 1989, similarities in the EPR and UV-vis absorption spectra for the blue multi-copper oxidases including laccase and ceruloplasmin had been observed [14] and a number of general conclusions made for the copper centers in ceruloplasmin as shown in Table 1 [13,15]. It was known that six copper atoms were nondialyzable and not available to chelation directly by dithiocarbamate and these coppers were assumed to be tightly bound and/or buried in the protein. Two of the coppers have absorbance maxima around 610 nm and these were interpreted as blue type I coppers with cysteine and histidine ligands, and responsible for the pronounced color of the protein. However, they are not equivalent and one of them, thought to be involved in enzymatic activity, is reduced and reoxidized at a faster rate than the second (e.g. see ref. 16). There was general concurrence that there are two type HI... 

Colorimetric, enzymatic, and immunochemical methods are available for the determination of ceruloplasmin. 

These observations underline the need for meticulous standardization of enz3Tnatic assay techniques for determining ceruloplasmin. With proper standardization, the enzymatic methods are reliable. However, one must always be alert to the possibility that factors other than the concentration of ceruloplasmin may influence the p-phenylenediamine oxidase activity. 

Immunochemical methods are also available for quantitative determination of ceruloplasmin (Hll, Mil, S15). These require a highly purified preparation of human ceruloplasmin, which serves as antigen and is given parenterally to another species, usually rabbits, to incite the production of specific antibody against the human ceruloplasmin. The specificity of the antibody depends entirely on the purity of the antigen. Further purification of the antibody can be achieved by absorption on sera devoid of ceruloplasmin, e.g., sera of certain patients with Wilson s disease. The immvmochemical method of determination of ceruloplasmin yields results which correlate well with serum copper levels and with the results of the enzymatic method. 

When conventional enzymatic assays are used to determine SOD activities in tissues, however, nonspecific reactions due to other proteins or low-molecular-weight compounds possessing SOD-like activities in the tissues can cause erroneous results. For example, serum ceruloplasmin reacts stoichiometrically with 02, though the reaction is noncatalytic under physiological conditions (B2,G10, HI,VI). Immunochemical assays for SODs are more reliable because the molecular weights and subunit structures of Cu,Zn-SOD and Mn-SOD are quite different from each other, and immunochemically these proteins do not cross-react. 

Ceruloplasmin is a blue a-2 glycoprotein of 132 kD. This plasma protein is responsible for the binding of 90 to 95% of the blood plasma copper in vertebrates. In addition to its primary role in copper transport and homeostasis, it possesses a number of additional enzymatic activities (see ref. 179 for a recent review). The complete amino acid sequence of human ceruloplasmin has been determined (42), establishing that this large multicopper oxidase is synthesized as a single chain polypeptide, containing 1046 amino acid residues. 

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