Subunit Characteristic Analysis Of The R-phycoerythrins From Marine Red Algae And Investigation On Their Chemical Stabilization

R-phycoerythrins (R-PEs) were extracted from two marine red macroalgaHeterosiphonia japonica and Polysiphonia urceolata in50mmol/L phosphate buffer.After ultrafiltration, the R-PEs of H. japonica and P. urceolata were respectivelyisolated from the extracts by the gel filtrations on Sephadex G-200and SephadexG-150, and then they were further purified and prepared by the ion exchangechromatography on DEAE-Sepharose FF. The R-PE of H. japonica, named HR-PE,showed isoelectric point (pI)4.6and the R-PE of P. urceolata, named PR-PE, showedpI4.5. The subunit analysis of HR-PE and PR-PE by SDS-PAGE and denaturingisoelectric focusing (denaturing-IEF) showed that the subunit bands showed in theSDS-PAGE were fewer than those showed in the denaturing-IEF. The results fromtwo dimension electrophoresis (2D-PAGE) demonstrated that the single band in thedenaturing-IEF may be separated into one more bands different in molecular mass bythe second phase of2D-PAGE and that the subunits with the same molecular massshowed in SDS-PAGE may show different pI in denaturing-IEF. This indicated thatamong the subunit components of HR-PE and PR-PE, the same type of the subunitswith equal molecular mass may have different pI, and the subunits which show thesame pI may have different molecular mass.The experimental results of the chemical stabilization of HR-PE and PR-PE withformaldehyde and EDC (1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimidehydrochloride) demonstrated that:1) the suitable reaction proportion of formaldehydeto HR-PE was212.31.0and the suitable reaction concentration of HR-PE was0.5310mg/ml. For PR-PE the suitable concentration was1.11mg/ml and the reactionproportion was54.71.0;2) the chemical reaction concentrations of HR-PE and EDCwere respectively3.0mg/ml and3.0m mol/L, and those of PR-PE and EDC were3.0mg/ml and5.0mmol/L, respectively. Compared with native HR-PE and PR-PE, thetolerant degree of the HR-PE and PR-PE chemically stabilized by formaldehyde andEDC to temperature,8mol/L urea,4%and8%SDS was raised obviously, and thestability of the formaldehyde-stabilized products was more than that ofEDC-stabilized ones. By means of bifunctional cross-linkers, the chemicallystabilized HR-PEs and PR-PEs can be covalently conjugated with immunoglobins.