Metalloproteins isolation

The plant lectin concanavalin A, a metalloprotein isolated from the jack bean, has also received considerable attention. (588-590,630,631) Using the stopped flow NMR technique three distinct conformation states of the protein, when it is bound to Mn, Ca, and a-methyl-D-mannoside, (630) have been deduced. 

CE is a useful tool for monitoring the purity of metalloproteins isolated from either natural or recombinant sources. CZE was used to follow the purification progress of metallothioneins in samples subjected to gel filtration chromatography and reversed-phase high-performance liquid chromatography (HPLC). " Detection of a unique chromophore arising from the interaction of metal ions... 

Isolation and purification of the metalloprotein. For instance, Hb was first crystallized in 1849, its physiological purpose of oxygen transport was recognized by 1864, and its molecular weight and primary amino acid sequence was known by 1930. 

A useful concept for the classification of metal-containing proteins has been suggested by Vallee The proteins are divided into two groups metalloproteins and metal-protein complexes, on the basis of their stability during the isolation procedures. Metalloproteins retain their metal constituent during fractionation and there is a stoichiometric relationship between the metal and protein. On the other hand, the metal is loosely bound and easily lost during dialysis in metal-proteins. Examples of both types of proteins can be found in an article by Vallee and Coleman... 

Bioavailable metals and metalloid species are either adsorbed or incorporated into the structure of proteins, lipids, nucleic acids, amino acids, sugars, vitamins and hormones to form complexes of varying degrees of thermodynamic stability and reactivity. These complexes could be classified as either metal-proteins or metalloproteins on the basis of their stability during isolation and purification (Vallee and Coleman, 1964). Whereas metal-proteins are relatively labile and the metal is easily lost during dialysis, metalloproteins are stable and inert. 

For GAOX, structural analysis is particularly complicated, because of the existence of multiple states of the enzyme differing essentially only in the number of electrons, i.e., the oxidation state of the metalloprotein complex. Three distinct oxidation states can be prepared, each with properties and reactivities dramatically different from the others, as indicated in Fig. 10 (Whittaker and Whittaker, 1988). When isolated from culture medium, GAOX is a mixture of two of these states a blue, one-electron reduced, catalytically inactive form (lAGO) that contains a Cu(II) ion and no radical and a green form that is catalytically active (AGO) and contains both Cu(II) and a free radical. The enzyme may be converted to... 

The turning point in the structural studies came after the work by Hiraoka et al. (1980) revealed that a-lactalbumin is a metalloprotein in which calcium is strongly bound (see also Sections VI and VII). Soon afterward three new crystal forms of bovine a-lactalbumin were isolated by Fenna (1982a), particularly trigonal Form II. Fenna (1982b) also isolated calcium-containing crystals of human a-lactalbumin suitable for X-ray structural analyses. The various crystalline forms of a-lactalbumin are summarized in Table III. 

Ceruloplasmin, the copper protein in plasma, deserves special attention. Human plasma contains approximately 32 mg/100 ml of this protein (see Table 6) (C2, C12, R4). According to the recent careful measurements of Kasper and Deutsch (K3), the molecular weight of ceruloplasmin is 160,000, somewhat higher than the 151,000 obtained by Pedersen (P3). Ceruloplasmin is an a2-globulin and contains 8 atoms of copper per molecule (H14). In addition to being a metalloprotein, it is a glycoprotein containing 7% carbohydrate hexose, hexosamine, and neuraminic acid (L4). Ceruloplasmin has been prepared in a pure form from human and pig sera (H14). Several newer methods are now available for its isolation and purification (B29, C20, D4, L4, S4, S7, S39). 

UEA I has been isolated by affinity chromatography on immobilized L-fucose columns [215]. Although originally believed to be a dimer of two dissimilar polypeptide chains, it has been shown to be composed of two identieal subunits containing 243 amino acid residues (Mr = 26 669 Da) held together by non-covalent interactions. The lectin is a metalloprotein requiring Ca and Zn " or Mn " for aetivity. 

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