Purification and autolysis of the ficin isoforms from fig (Ficus carica cv. Sabz) latex

Abstract

Ficin (EC 3.4.22.3), a cysteine endoproteolytic protease in fig trees' latex, has multiple isoforms. Until now, no data on autolysis of individual ficins (ficin isoforms) are available. Following purification, ficins' autolysis was determined by HPLC chromatogram changes and ultrafiltrations at different temperatures and storage times. These results showed that the number of HPLC peaks in latex proteins purification of Ficus carica cv. Sabz varied from previous fig varieties or cultivars. Proteolytic activity of ficins was inhibited by specific cysteine protease inhibitors, confirming the participation of the cysteine residue in the active site. The zeta potential of the first two eluted peaks (I and II) was negative, while that of other peaks were positive. All ficins were susceptible to autolysis when stored at high temperatures. In contrast, only the last two ficins (B, C) were prone to autolysis at cold temperature after long storage period. The rate of degradation of the ficins was significantly increased with the increased storage time. The ficin (A) related to peak (III) had the highest and the lowest surface hydrophobic patches and ratio of autolytic to proteolytic activity, respectively.

Fig. 1

Chromatographic separation of ficins from Ficus carica cv. Sabz latex on HPLC using a SP-Sepharose high-performance column. The column was equilibrated with 0.01 M sodium phosphate buffer at pH 7.0. Then it was eluted using gradient of 0.3–0.6 M NaCl (·········) in the same buffer, and the eluted fractions were analyzed by absorbance measurements at 280 nm (—●—) and caseolytic activity (units/ml enzyme) (—◇—).

Fig. 2

Inhibitory activity measurements of the UF, RF1 and ficins related to HPLC chromatogram peaks (I, II, III, IV, V and VI). Activity in presence of bovine casein substrate without inhibitor (□), activity in the presence of potassium tetrathionate ( ) and iodoacetamide (■) inhibitor.

Fig. 3

Assessment of the homogeneity of the purified ficin related to HPLC chromatogram peak (III) by (a) SDS–PAGE pattern using 15% gel. Lane 1, molecular-mass markers; lane 2, purified ficin; (b) gel filtration using Superdex 75 column; and (c) re-chromatography using SP-Sepharose column.

Fig. 4

These bar graphs show the results of Zeta potential measurements for UF, RF1 and ficins related to HPLC chromatogram peaks in order of their elution time from cation-exchange column. UF related to peak (I) is before and the rest are after salt gradient elution.

Fig. 5

Effects of storage time on the autolysis of UF and ficins related to HPLC chromatogram peaks (I), a; (III), b; (IV), c; and (VI), d; with period (■) 1, (□) 5, and ( ) 10 days storage by using ultrafiltration membranes in absence of substrate and inhibitor.

Fig. 6

Chromatogram profile before ( ) and after (·········) incubation at 37 °C for 24 h. When the incubated solution was subjected to cation exchange column, the main peak of the elution pattern, coinciding with the band of native ficin, was forwarded by one peptide peak produced or increased from autolysis of the UF and ficins related to HPLC chromatogram peaks; (I), a; (III), b; (IV), c; and (V), d in absence of substrate and inhibitor.

Fig. 7

Chromatogram profiles changes after incubation at 4 °C for 20 days. When the autolyzed solution was subjected to cation exchange column, the main peak of the elution pattern, coinciding with the band of native ficin, was forwarded by several peptide peak produced from autolysis of ficin related to HPLC chromatogram peaks; (IV), a; and (V), b in absence of substrate and inhibitor.

Fig. 8

Autolytic activity (% produced peptides from autolysis/UF or ficin used before autolysis) of the UF and three ficins related to HPLC chromatogram peaks (I, III, IV and V) at 37 °C for 24 h in absence of substrate and inhibitor.

Fig. 9

Extrinsic (ANS) fluorescence spectra of the UF and three ficins related to HPLC chromatogram peaks; I, (·········); III, (—); IV, ( ); and V, ( ).

Fig. 10

Correlation between zeta potential modulus (■), surface hydrophobic patches factor ( ), and ratio of autolytic activity to proteolytic activity (□) of the UF and three ficin related to HPLC chromatogram peaks (I, III, IV and V).