Mj. Hubbard, RAPID PURIFICATION AND DIRECT MICROASSAY OF CALBINDIN9KDA UTILIZING ITS SOLUBILITY IN PERCHLORIC-ACID, Biochemical journal, 293, 1993, pp. 223-227
The 9 kDa calcium-binding protein, calbindin9kDa, was found to be solu
ble in 7% (v/v) perchloric acid. Calbindin9kDa was easily purified fro
m 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 o
f tissue (mean +/- S.E.M.; n = 3) from normally fed 7-8-week-old rats
(approx. 70% recovery). The purification was also effective with rabbi
t duodenum calbindin9kDa, but not with various other EF-hand calcium-b
inding proteins tested in the rat. Several criteria (h.p.l.c., u.v. sp
ectrum, denaturing two-dimensional PAGE, N-terminal sequencing) indica
ted that the rat calbindin9kDa was purified to homogeneity and was not
affected by proteolysis. High-affinity calcium-binding properties wer
e retained and no evidence of isoforms or charge modification was obse
rved. 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).