DETECTION AND PURIFICATION OF MODIFIED LEGHEMOGLOBINS FROM SOYBEAN ROOT-NODULES

A new procedure was developed to purify leghemoglobins (Lbs) from Glyc ine max L. (Merr). root nodules. This procedure resulted in the purifi cation of a hemoprotein fraction, Lbam. Lbam as detected by ion-exchan ge-HPLC was found in nodule extracts prepared in the presence of pepti dase inhibitors, and was unstable in acidic solutions. Acidic conditio ns also enhanced proteolysis of Lba by a nodule fraction enriched in p eptidases as observed using a denaturing peptide gel developed for thi s purpose. In contrast, there was little degradation of Lba by endogen ous peptidases at pH 8.0. We incorporated these observations into the purification protocol as follows. (1) nodule extracts were first chrom atographed on hydroxylapatite at pH 6.8. Greater than 98% of the pepti dases bound to the column and the Lbs were recovered in the wash fract ion; (2) subsequent ion-exchange chromatography of the wash fraction a t pH 8.0 yielded several fractions related to Lba, including Lbam; and (3) The crude Lbam fraction was resolved into four distinct protein b ands by isoelectric focusing using a new buffer system and gel formula tion. One of these bands exhibited a pI value identical to that of Lba , whereas the other three proteins displayed more acidic pI 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 c ompositions and possessed a sequence identical to that of Lba in the f irst 15 residues from the amino-terminal end.