Protein-Metal Complexes

Table 9 gives some cases where the rotational strengths of absorption bands have been measured in metalloproteins. At the present time these changes are not used to diagnose the nature of the ligands of the metal but rather they have been used to follow minor changes at the metal when substrates or inhibitors interact with the metals. The sensitivity of CD and MCD measurements to very small changes in the metal environment make them very attractive for protein/metal complex studies. 

A complete systematic description of protein-metal complexation has yet to be presented, but it is apparent that many mechanisms are involved. Some proteins may participate in classical chelation interactions via polycarboxy clusters on their surfaces.2 Others interact with metals via coordination with polyhis-tidyl or other aromatic domains.13 5 Still others may interact with metals via sulfhydryl residues.13 The literature on immobilized metal affinity reveals examples of unexplained retention that may involve yet other mechanisms.1... 

Although a-D-mannosidase from mammalian, plant, and molluscan sources is dependent upon zinc for its catalytic activity, the addition of this ion has a marked effect in the enzyme assay only at those pH values where the active, protein-metal complex dissociates appreciably despite the presence of substrate. (Dissociation, which is greater at lower values of pH, is lessened in the presence of substrate.) The presence of zinc ion in the assay (0.1 mM) is thus of particular importance in the case of the limpet enzyme, where the pH of optimal activity is 3.5. Jack-bean and rat-epididymal a-D-mannosidase are both assayed at pH 5, and up to 10% activation may be observed with zinc. 

At pH values above neutrality, jack-bean a-D-mannosidase, unlike the enzyme from the other two sources, is stable, and the native, protein-metal complex does not dissociate. By working at pH 8, it is possible to purify the enzyme from jack-bean meal without addition of zinc.27 The procedure shown in Table V was followed with only slight modification (see Table IX, Section III,5 p. 433). 

The position with regard to the limpet enzyme is more complicated.46 Although zinc has little effect in the assay at pH 5, this pH is so far removed from the sharp optimum at pH 3.5 (Cl- present) that the enzyme displayed less than 20% of its activity. Furthermore, a large proportion of the enzyme seemed to be firmly complexed with toxic cations derived from the limpet, and these were not displaced by addition of an excess of zinc during an assay at pH 5 (see Fig. 1). At its relatively low pH of optimal activity, the limpet protein-metal complex was readily dissociable, even in the presence of substrate, and, consequently, on assay at this pH, Zn2+ seemed to displace toxic cations, allowing the enzyme to display its full, potential activity. 

One of the most striking indications of the importance of Zn2+ for a-D-mannosidase activity was obtained with preparations that had been inactivated by incubation with EDTA. On addition of an excess of Zn2+ to the assay mixture, complete activity was regained instantaneously, regardless of the extent of prior inactivation. (When the EDTA-inactivated enzyme described in Fig. 4 was assayed in the presence of Zn2+, the points followed the line for the Zn2+-stabilized enzyme.) Again, no other cation that we have examined can replace Zn2+, leaving little doubt as to the identity of the activating cation in the original material. It also follows that EDTA must withdraw Zn2+ from the protein-metal complex. Had EDTA merely formed a... 

Substrate not only arrested spontaneous inactivation of the enzyme, but prevented the restoration of activity by Zn2+ in a preparation that had already undergone spontaneous decay. It was concluded that, regardless of whether it contains Zn2+ or a toxic cation, the substrate so combines with the protein-metal complex as to prevent dissociation, and, hence, inactivation or reactivation as the case might be. On the other hand, the addition of Zn2+ caused immediate reactivation of an EDTA-treated preparation, even in the presence of substrate, suggesting that the substrate cannot combine with the metal-free, enzyme protein. 

One of the most powerful separation techniques available is high-performance liquid chromatography (HPLC) [6], It has a broad range of applicability which also encompasses non-volatile substances such as ionic compounds (e.g. amino acids, proteins, metal complexes) or high-molecular weight compounds, such as... 

An important property of the protein metal complexes is that their solubilities are particularly influenced by the dielectric constant of the medium. Thus, a moderate reduction of the dielectric constant of an aqueous solution will precipitate the Ba or Zn salts of many proteins which are readily soluble in the aqueous solutions. This property has been used for the fractionation of the plasma proteins. The use of barium and zinc salts not only gave superior fractionation, but also allowed the use of lower ethanol concentrations with diminished risk of denaturation. 

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