Identification of a novel copper-binding protein from the liver of  Indian childhood cirrhosis: purification and physicochemical characterization [corrected].
A novel copper-binding protein was identified in the liver supernatant (100,000 x g) of Indian childhood cirrhosis (ICC),  purified to apparent homogeneity and characterized [corrected]. Purified major copper-binding protein (MCuBP) is solely responsible for binding about 35% of the total supernatant copper. Elution profile of ICC liver supernatant on Sephadex G-75 column chromatography showed three peaks. About 60% of the total supernatant copper was resolved in peak II, whereas zinc content was insignificant in this peak. But peak II was almost missing in a gel elution profile of control liver supernatant. The control group included cases of various liver diseases viz. neonatal hepatitis, septicemia, and mixed nodular cirrhosis. Copper-binding proteins of peak II further purified on ion-exchange chromatography and elution profile showed that peak II was a MCuBP with high copper-binding capacity (10 g atoms/mol of native protein). SDS-PAGE of this protein also revealed the existence of a single band with molecular mass of about 50 kD. UV spectra of MCuBP showed the maximal absorbance at 254 nm. Unlike the classical metallothionein, the amino acid composition of MCuBP revealed the presence of aromatic amino acids and higher content of glutamic acid and aspartic acid followed by glycine and serine. The ratio (0.3) of basic amino acids to acidic amino acids strongly indicates that it is an acidic protein. The cysteine content in this protein was insignificant, which further corroborates the possibility that the acidic amino acids might be prominent candidates for binding copper. Thus, the 50-kD MCuBP apparently makes a major contribution to the total copper-binding activity in ICC liver cytosol and may play a significant role in hepatic intracellular copper accumulation.
Prasad
R
R
Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India.
Kaur
G
G
Mond
R
R
Walia
B N
BN
D000596:Amino Acids / Q000032:analysis; D002352:Carrier Proteins / Q000737:chemistry / Q000302:isolation & purification*; D002675:Child, Preschool; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000378:metabolism*; D006801:Humans; D007194:India; D008103:Liver Cirrhosis / Q000378:metabolism*; D008297:Male; D008970:Molecular Weight; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet; D015032:Zinc / Q000378:metabolism
D000596:Amino Acids; D002352:Carrier Proteins; C023548:copper-binding protein; D003300:Copper; D015032:Zinc