Cold Steeping Infusion, a Novel Lectin Extraction Technique for the Isolation, Purification and Partial Characterization of Lectins from the Green Venezuelan Marine Alga Caulerpa serrulata

Abstract

A lectin from the green Venezuelan marine alga Caulerpa serrulata was extracted with phosphate buffered saline (PBS) using cold steeping infusion (CSI) and by grinding with liquid nitrogen (GLN). The proteins were precipitated using solid ammonium sulfate. Both the crude extracts and ammonium sulfate precipitated proteins were tested for hemagglutinins using native and papain-treated human red blood cell suspensions in isotonic saline solution. Purification of lectins was achieved using affinity chromatography-sugar-epoxy-sepharose 6B and molecular weight was assessed by size exclusion chromatography using Bio-gel® P-100 and SDS-PAGE with 2-mercaptoethanol. IEF-urea 8M was also evaluated. Using CSI it was shown that the marine alga released hemagglutinating compounds into the solutions; the same hemagglutinating compounds were also obtained by GLN. Ammonium sulfate precipitated proteins exhibited agglutinating activity against native and papain-treated human red blood cells. Temperature and EDTA were shown to affect dramatically the lectin activity towards red blood cells. A lectin was purified efficiently and the molecular weight calculated as approximately 78,000 Daltons. The CSI technique demonstrated that the alga could be returned to an active metabolic state by immersion in a simple buffer after having been kept dormant by freezing at −20°C for long periods. It was also shown that the alga was releasing bioactive compounds into the solutions and, therefore, this procedure is being suggested as a good, gentle, non-disruptive extraction technique and we postulate CSI as a possible bioreactor for the continuous production of bioactive compounds from green marine algae.