Metalloprotein analysis by capillary isoelectric focusing

Capillary isoelectric focusing (cIEF) was used to analyze three metalloproteins: conalbumin, transferrin and metallothionein (MT). Two different ampholyte mixtures were employed that generated linear pH gradients of 3–10 and 5–8. Several different proteins and one peptide known isoelectric points (pIs) were used to establish linear relationships between peak migration time and pI. These standards were also used as internal markers to estimate peak pI values of the metalloproteins subjected to cIEF. Conalbumin (iron-free) subjected to cIEF with a pH gradient of 3–10 yielded a single major component (pI 7.17). When the protein was saturated with iron (2 Fe³⁺/mol protein), a shift to lower pI was observed with a major peak (pI 6.24) and a lesser peak (pI 6.09). Mixing iron-free with iron-saturated conalbumin or adding iron to iron-free conalbumin prior to cIEF produced an additional peak (pI 6.68) that was presumed to be conalbumin containing a single iron atom (monoferric form). Human transferrin subjected to cIEF with a pH range of 3–10 gave a similar separation pattern to conalbumin with four major peaks at pI values of 6.25 (apotransferrin), 5.96 (monoferric form), 5.48 and 5.34 (differic forms). Additional resolution of the molecular forms of both conalbumin and transferrin was achieved using a narrower pH gradient (5–8). Rabbit liver MT subjected to cIEF with a pH gradient of 3–10 gave a complex separation pattern with two prominent peaks (pI values of 3.73 and 3.56) that were presumed to be the fully metal-saturated MT-1 and MT-2 isoforms. When individual MT isoforms (MT-1 and MT-2) were separately subjected to cIEF with a pH gradient of 3–10, heterogeneous peaks with higher pI values (4.12–4.74) were observed. In contrast, horse kidney MT gave a single predominant peak with a pI of 4.09. MT samples could be separated using pH gradient of 5–8 despite the fact that their apparent pI values were below the limits of the pH gradient established. In general, the heterogeneity observed for conalbumin, transferrin and MT proteins subjected to cIEF reflects the presence or absence of bound metal. Thus, cIEF represents a potentially useful analytical method which can provide information concerning the metal-binding characteristics of these and perhaps other metalloproteins.