환자의 혈장이나 복수로부터 C-reactive protein을 높은 수율로서 순수하게 분리정제할 수 있는 간이 정제법을 개발하였다. DPPC-Affinity chromatography와 Hydroxylapatite chromatography 분리과정을 거쳐 간단하고 신속한 방법으로 분리된 CRP는 단일확산 침전법과 폐렴균의 세포막인 C-polysaccharide를 이용한 EIA 방법으로 활성도를 측정하였다. 분리한 CRP는 SDS-PAGE에서 단일밴드로 나타났으며 immunoelectrophoresis에서도 단일 침전 arc로 나타나 순수분리 되었음을 확인하였다. 또한, Sephacryl S-200 gel filtration과 SDS-polyacrylamide gel 전기영동 분석결과에 의하면 분자량은 118,000으로서 5개의 동일한 subunit로 구성되어 있으며 그 subunit의외 분자량은 23,600이었다. CRP의 isoelectric point는 4.82였으며 500 ml의 복수로부터 분리된 CRP의 회수율은 약 67%로서 4.7 mg이었다.
A simplified procedure for obtaining high purity and high yield of C-reactive protein (CRP) was established. The CRP from ascitic and pleural fluid was purified using calcium dependent affinity chromatography of CNBr-activated Sepharose-4B covalently coupled to p-diazonium phenylphosphorylcholine (DPPC) and hydroxylapatite chromatography. The active fractions of CRP during the purification procedure were determined by EIA using pneumococcal C-polysaccharide. The final recovery of CRP was 67% of the initial protein amount as quantitated by single radial immunodiffusion (SRID), and the purification fold was 3,600. The purified CRP was shown to be homogeneous by electrophoresis and immunoelectrophoresis. The molecular weight of CRP was approximately 118KD as determined by Sephacryl S-200 gel filtration and the molecular weight of a subunit was approximately 23.6 KD by SDS-polyacrylamide gel electrophoresis. The isoelectric point of CRP was found to range about 4.82.
