Metalloproteins purification

Metal chelate affinity chromatography finds most prominent application in the affinity purification of recombinant proteins to which a histidine tag has been attached (described later). As protein binding occurs via the histidine residues, this technique is no more inherently useful for the purification of metalloproteins than for the purification of non-metalloproteins (a common misconception, given its name). 

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. 

Metal complexation — One of the most insidious and widely occurrent sources of analytical variation in IEC is product complexation with metal ions. Most proteins can form complexes with metals, regardless of whether or not they are metalloproteins.1 Participant metal ions can derive from the cell culture production process, purification process buffers, or even stainless steel chromatography systems. Complexation can alter retention times, create aberrant peaks, and substantially increase peak width. To the extent that metal contamination of your sample is uncontrolled, so too will be the performance of your assay. 

F. Schneider, Purification and partial charaderization of amino-acylase from Aspergillus oryzae, in Metalloproteins,... 

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. 

These concepts have proved themselves pragmatically useful and led to the identification of three metalloproteins (Vallee, Hoch, and Hughes, 1954 Vallee and Neurath, 1954 Vallee and Hoch, 1955a,b). In all instances, the firm association of the metal made it possible to follow the course of purification of the protein by metal analyses. 

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). 

The peanut Arachis hypogaea) contains a lectin with anti-T (Gal(Pl-3)GalNAc) activity [174]. This antigen appears on human erythrocytes following treatment with sialidase and leads to the phenomenon known as polyagglutinability as monitored by the peanut lectin [175]. Peanut agglutinin, purified by numerous affinity purification schemes, is a tetrameric protein composed of four carbohydrate-free subunits, A/f = 27000Da[176]. The lectin is a metalloprotein rich in acidic and hydroxylic amino acids and devoid of cysteine [176]. 

Enzyme Concentration and Purification. The number of existing enzymes is estimated to be more than 10,000 of which more than 100 have been purified in crystalline form and over 600 in fairly purified form. The molecular weight of enzymes varies from 12,700 (ribonuclease) to over 1,000,000 (L-glutamate dehydrogenase, d-carboxylase). All enzymes are proteins, conjugated proteins or metalloproteins containing one or more active sites per molecule. 

According to Vallee and Coleman (1964) the association between metals and proteins can be divided into two groups namely metalloproteins and metal-protein complexes. In metalloproteins the metal forms an integral part of the protein structure, and it is present in stoichiometric amounts. Furthermore, the metal is not lost during the purification step. The transition metals have a tendency to form this type of association. These complexes have high thermodynamic stability and are relatively inert. On the other hand, in metal-protein... 

Purification of Zinc-Containing Ferredoxin from Sulfolobus sp. Strain 7. The isolation of ferredoxin from the aerobic thermoacidophilic crenarchaeote, Sulfolobus acidocaldarius, was described by Kerscher et al. This procedure was adapted to purify ferredoxin and related metalloproteins from the Sulfolobus sp. strain 7 cells. 

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... 

Immobilized metal-affinity chromatography (IMAC) is also known as metal-chelate affinity chromatography (MCAC). This method was first proposed by Porath et al. in 1975 [63] and is based on the specific interactions between immobilized metal ions and amino acid residues, such as histidine, fiyptophan, and cysteine in proteins or peptides [63]. IMAC has become an important tool for the detection and purification of metalloproteins, histidine-tagged proteins, and phosphorylated proteins. Areas in which this method is now used include proteomics [64—66], work with recombinant proteins [67—69], and disease diagnosis [70,71]. 

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