The proteins of Hevea brasiliensis latex. 4. Isolation and characterization of crystalline hevein

Fresh rubber latex obtained from Hevea bra8iliensis consists of a suspension of several types of particles in an aqueous serum, and can be separated into fractions by high-speed centrifuging (Cook & Sekhar, 1953).The top layer in the centrifuge tube consists mainly of rubber particles, below which is a layer of almost clear serum.At the bottom of the tube is a layer referred to in this paper as the 'bottom fraction', which contains sedimentable bodies including the lutoid particles described by Homans & van Gils (1948).In preliminary work an aqueous extract of the bottom fraction was found to contain several electrophoretically distinct protein components.This observation has been confirmed and elaborated by Moir & Tata (1960).This paper describes the isolation and characterization of a major component which is anionic at pH 8-6 and for which the name hevein is proposed.EXPERIMENTAL Materials.Freeze-dried samples of the bottom fraction of freshly tapped latex were used throughout.Collection of the latex and centrifugal separation and freeze-drying of the bottom fraction were carried out by the staff of the Rubber Research Institute of Malaya, as described by McMullen (1959).All freeze-dried fractions were sealed under high vacuum in Malaya, and on arrival at our Laboratory were stored at -150 until required.Elementary analyses.Total N was determined by the micro-Kjeldahl method and C, H and ash by Pregl micro- combustion.The S content of the ash and gaseous com- bustion products was measured turbidimetrically as BaSO4 after complete dissolution of the ash in dilute HC1.Where the amount of material available for analysis was limited, and the ash content was known to be low (as in purified hevein), C, H and S were determined on the same sample with a microcombustion tube containing a roll of silver and PbO2 only.P was determined colorimetrically with ammonium molybdate after wet oxidation.Soluble proteins were extracted from the freeze-dried solids with 0*05M-phosphate buffer, pH 6-8, or with 0-05 m- Na2B407, pH 9-3.The protein-N content of the extract was then determined by tannic acid precipitation and Kjeldahl analysis (Archer & Sekhar, 1955).Purifiedpro- tein samples were dried to constant weight in vacuo at 800 before analysis.