Rapid purification and direct microassay of calbindin9kDa utilizing its solubility in perchloric acid.
The 9 kDa calcium-binding protein, calbindin9kDa, was found to be soluble in 7% (v/v) perchloric acid. Calbindin9kDa was easily purified from rat duodenum in 1 day with perchloric acid precipitation followed by reverse-phase h.p.l.c. The yield was 21.4 +/- 2.3 nmol/g wet weight of tissue (mean +/- S.E.M.; n = 3) from normally fed 7-8-week-old rats (approx. 70% recovery). The purification was also effective with rabbit duodenum calbindin9kDa, but not with various other EF-hand calcium-binding proteins tested in the rat. Several criteria (h.p.l.c., u.v. spectrum, denaturing two-dimensional PAGE, N-terminal sequencing) indicated that the rat calbindin9kDa was purified to homogeneity and was not affected by proteolysis. High-affinity calcium-binding properties were retained and no evidence of isoforms or charge modification was observed. Residue 59, identified as Asn (not Asp as previously reported), was fully amidated. When adopted as a microassay with isocratic h.p.l.c., the perchloric acid procedure enabled rapid (less than 6 min) and direct (peptide bond absorbance) quantification of less than 1 pmol of calbindin9kDa. This new approach to purification and assay will be of particular utility for investigations of calbindin9kDa in previously intractable low-abundance sources (e.g. cultured cells).
Hubbard
M J
MJ
Department of Biochemistry, University of Otago, Dunedin, New Zealand.
D000595:Amino Acid Sequence; D000818:Animals; D064026:Calbindins; D002851:Chromatography, High Pressure Liquid; D004386:Duodenum / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010472:Perchlorates / Q000737:chemistry*; D011817:Rabbits; D051381:Rats; D017208:Rats, Wistar; D064030:S100 Calcium Binding Protein G / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility; D013056:Spectrophotometry, Ultraviolet
D064026:Calbindins; D010472:Perchlorates; D064030:S100 Calcium Binding Protein G