Ceruloplasmin separation

The specific interaction of Cibacron Blue and its derivatives to dinucleotides, mainly to NAD, NADP and ATP offers the possibility of purifing all enzymes which are dependent on these coenzymes. According to Mosbach < - > there are 163 enzymes requiring NAD, 141 enzymes requiring NADP, about 40 enzymes requiring NADP or NAD and 225 enzymes dependent on ATP. Besides these specific interactions non-specific interactions of Cibacron Blue and its derivatives with. proteins can also be applied for separation purposes. The non-specific interaction of Cibacron Blue with human serum albumin, for instance, enables albumin to be removed from transferrin, ceruloplasmin or other plasma proteins in order to purify human... 

Figure 5 depicts correlations between serum ceruloplasmin and copper concentration as can be seen, it is significant in both males and females with p < 0.005, the correlation coeflBcients being 0.36 and 0.84, respectively. However, when different lots of immunodiffusion plates were used and correlation coeflBcients for the samples welre calculated separately, the range of correlation in five lots was 0.30-0.88, with a mean value of 0.53. 

CHAPTER 36, FIGURE 6 Cofactor proteins, factor V and factor VIII. Factor V and factor VIII coagulant (not the von Willebrand factor carrier of factor VIII) contain six distinct structural domains. The two A domains. A, and Aj at the N-terminal end of the polypeptide chain, are separated from the Aj domain by a highly glycosylated B domain. The two C domains are at the C-terminal end of the molecule. The A domain sequences are homologous to the A domains of ceruloplasmin. Both factor Va and factor Villa act as catalysts in the activation of prothrombin and factor X, respectively. Activation sites are indicated by green arrows inactivation sites by red arrows. In factor Va, complete inactivation requires cleavage of Arg ° . 

Hypocupremia and low blood levels of ceruloplasmin are one of the most characteristic biochemical alterations in Wilson s disease. This condition is discussed in a separate section below. 

B28. Broman, L., Separation and characterization of two ceruloplasmins from human serum. Nature 182, 1655-1657 (1958). 

Human ceruloplasmin consists of a single polypeptide chain with a MW of 132 kDa folded in six cupredoxin domains arranged in a triangular array. Each domain comprises a p-barrel, constructed in a Greek key motif, typical for the cupredoxins. Three of the six copper ions are bound to T1 sites present in domains 2, 4, and 6, whereas the other three copper ions form a trinuclear cluster, bound at the interface between domains 1 and 6 (Fig. 10). The spatial relation between the trinuclear center and the nearest T1 site (A, in domain 6) closely resembles that found in AO and was taken to further support the proposal that hCp has an oxidase function. The three T1 sites are separated from each other by a distance of 1.8 nm, a distance that might still allow for internal ET at reasonable rates and could also increase the probability for electron uptake. The coordination sphere of the T1 site in domain 4 (TIB) is identical with that of domain 6 (TIA). The third type 1 center (TIC), however. 

Normal human serum actually contains two chromatographically distinct forms of ceruloplasmin. A calcium phosphate column cleanly separates major (I) and minor (II) components, the latter being approximately 15% of the total ceruloplasmin present in the serum (109—111). Ryden (112, 113) has shown that the two forms though identical in amino acid composition and Cu binding capacity, differ in the degree to which carbohydrate components have been affixed to the protein during biosynthesis. Most of the presently accepted physical properties of human ceruloplasmin are presented in Table 2. 

IgD, ceruloplasmin, / 2-glycoprotein I, hemopexin, and leucine-rich glycoprotein are shown in Fig. 3. Ceruloplasmin, which is a multicopper oxidase whose amino acid sequence is known to be homologous to that of coagulation factor VIII, was separated into three major peaks by hydroxyapatite HPLC. The first peak does not have absorbance at 610 nm therefore, it contains apoceruloplasmin that does not have enzyme activity. The second peak (type 1) contains four glucosamine... 

Hydrophobic interaction columns separate proteins using hydrophobicity, similar to that of reversed-phase HPLC. However, this method provides an alternative approach with little or no denaturation of proteins because organic solvents are not required for the elution. In fact, ceruloplasmin, which was denatured during reversed-phase chromatography could be eluted from a hydrophobic interaction column without denaturation (Fig. 6). /f2-Glycoprotcin I, which has been... 

---