Detection and purification of modified leghemoglobins from soybean root nodules

Abstract

A new procedure was developed to purify leghemoglobins (Lbs) from Glycine max L. (Merr). root nodules. This procedure resulted in the purification of a hemoprotein fraction, Lbam as detected by ion-exchange-HPLC was found in nodule extracts prepared in the presence of peptidase inhibitors, and was unstable in acidic solutions. Acidic conditions also emhanced proteolysis of Lba by a nodule enriched in peptidases as observed using a denaturing peptide gel developed for this purpose. In contrast, there was little degradation of Lba by endogenous peptidases at pH 8.0. We incorporated these observations into the purification protocol as follows: (1) nodule extracts were first chromatographed on hydroxylapatite at pH 6.8. Greater than 98% of the peptidases bound to the column and the Lbs were recovered in the wash fraction; (2) subsequent ion-exchange chromatography of the wash fraction at pH 8.0 yielded several fractions related to Lba, including Lbam; and (3) The crude Lbam fraction was resolved into four distinct protein bands by isoelectric focusing using a new buffer system and gel formulation. One of these bands exhibited a p I value identical to that of Lba, whereas the other three proteins displayed more acidic p I values of 4.91, 4.87 and 4.85, respectively. All of these purified Lbam proteins had molecular weights indistinguishable from Lba, similar amino acid compositions and possessed a sequence identical to that of Lba in the first 15 residues from the amino-terminal end.