임상 치료제인 알부민을 정제하여 알부민의 중합체형성과 변성인자에 의한 기작을 연구하고, 알부민과 세룰로플라스민 결합과 세룰로플라스민의 역할을 생화학적으로 연구하였다. 그리고 알부민 및 세룰로플라스민의 정제방법을 제시하였다. 알부민은 냉 에탄을 분획법, DEAE-Sepharose fast flow ...
임상 치료제인 알부민을 정제하여 알부민의 중합체형성과 변성인자에 의한 기작을 연구하고, 알부민과 세룰로플라스민 결합과 세룰로플라스민의 역할을 생화학적으로 연구하였다. 그리고 알부민 및 세룰로플라스민의 정제방법을 제시하였다. 알부민은 냉 에탄을 분획법, DEAE-Sepharose fast flow크로마토그래피로 정제하였으며, HPLC로 분석하고, 역류면역전기영동에 의해 알부민과 이종 단백질인 세룰로플라스민, 합토글로빈, a-l-lipoprotein, 알부민 전구체를 확인하였다. 알부민을 pH 7.0에서 처리하고, 60℃에서 10시간 열처리 한 결과 총 지질함량이 감소되어 pH 4.6 조건보다 알부민 용해능이 우수하게 나타났다. HPLC와 FPLC 결과 열처리 전에는 알부민이 이합체 형태로 나타났으며 열 처리 후에는 중합체가 증가되었다. 알부민을 SDS-PAGE 한 결과 열처리 전, pH 4.6, 또는 pH 7.0 의 조건에서는 합토글로빈이 확인되었으나 열처리 후에 나타나지 않았다. 합토글로빈은 열처리 과정에서 알부민을 변성시키는 단백질로 확인되었다. 또한 0.22㎛ 제거법은 알부민 이합체와 중합체 제거에는 적합하지 않았다. 알부민을 2년간 보존 시 pH가 7.0에서 6.8로 느리게 변화되었고 탁도는 거의 변화되지 않아 보존기간에 따른 변성은 없는 것으로 나타났다. 그러나 헬 함량은 서서히 증가되어 1년 이상 보존 시 색의 변화를 나타냈다. 알부민은 DEAE-Sephacel과 Prep-Cell에 의해 순수정제 되었고, 세룰로플라스민은 QAE-Sephadex A-50과 DEAE-Sepharose CL-6B에 의해 정제 되었다. 알부민과 세룰로플라스민은 oxidase 활성이 나타났다. 세룰로플라스민 및 알부민은 열처리하고 안정제를 첨가한 경우 oxidase 활성이 감소되었으며, 안정제가 세룰로플라스민의 활성을 억제하는 것으로 나타났다. 알부민에 세룰로플라스민을 가한 용액의 ferroxidase 활성은 열처리하고 안정제를 첨가한 용액에서 증가되어 oxidase 활성과는 반대의 결과를 나타냈다. 안정제가 ferroxidase 활성을 억제하고, 열처리한 후에만 ferroxidase 활성을 증가시켰다. 그러므로 알부민에서 세룰로플라스민의 분리는 온도와 관련이 있는 것으로 나타났다. 알부민의 보존기간에 대한 ferroxidase 활성은 감소되었다. 따라서 생체내에서 알부민과 세룰로플라스민은 서로 결합하는 단백질로 항산화 기능을 하는 것으로 사료된다.
임상 치료제인 알부민을 정제하여 알부민의 중합체형성과 변성인자에 의한 기작을 연구하고, 알부민과 세룰로플라스민 결합과 세룰로플라스민의 역할을 생화학적으로 연구하였다. 그리고 알부민 및 세룰로플라스민의 정제방법을 제시하였다. 알부민은 냉 에탄을 분획법, DEAE-Sepharose fast flow 크로마토그래피로 정제하였으며, HPLC로 분석하고, 역류면역전기영동에 의해 알부민과 이종 단백질인 세룰로플라스민, 합토글로빈, a-l-lipoprotein, 알부민 전구체를 확인하였다. 알부민을 pH 7.0에서 처리하고, 60℃에서 10시간 열처리 한 결과 총 지질함량이 감소되어 pH 4.6 조건보다 알부민 용해능이 우수하게 나타났다. HPLC와 FPLC 결과 열처리 전에는 알부민이 이합체 형태로 나타났으며 열 처리 후에는 중합체가 증가되었다. 알부민을 SDS-PAGE 한 결과 열처리 전, pH 4.6, 또는 pH 7.0 의 조건에서는 합토글로빈이 확인되었으나 열처리 후에 나타나지 않았다. 합토글로빈은 열처리 과정에서 알부민을 변성시키는 단백질로 확인되었다. 또한 0.22㎛ 제거법은 알부민 이합체와 중합체 제거에는 적합하지 않았다. 알부민을 2년간 보존 시 pH가 7.0에서 6.8로 느리게 변화되었고 탁도는 거의 변화되지 않아 보존기간에 따른 변성은 없는 것으로 나타났다. 그러나 헬 함량은 서서히 증가되어 1년 이상 보존 시 색의 변화를 나타냈다. 알부민은 DEAE-Sephacel과 Prep-Cell에 의해 순수정제 되었고, 세룰로플라스민은 QAE-Sephadex A-50과 DEAE-Sepharose CL-6B에 의해 정제 되었다. 알부민과 세룰로플라스민은 oxidase 활성이 나타났다. 세룰로플라스민 및 알부민은 열처리하고 안정제를 첨가한 경우 oxidase 활성이 감소되었으며, 안정제가 세룰로플라스민의 활성을 억제하는 것으로 나타났다. 알부민에 세룰로플라스민을 가한 용액의 ferroxidase 활성은 열처리하고 안정제를 첨가한 용액에서 증가되어 oxidase 활성과는 반대의 결과를 나타냈다. 안정제가 ferroxidase 활성을 억제하고, 열처리한 후에만 ferroxidase 활성을 증가시켰다. 그러므로 알부민에서 세룰로플라스민의 분리는 온도와 관련이 있는 것으로 나타났다. 알부민의 보존기간에 대한 ferroxidase 활성은 감소되었다. 따라서 생체내에서 알부민과 세룰로플라스민은 서로 결합하는 단백질로 항산화 기능을 하는 것으로 사료된다.
Albumin is the protein of the highest concentration in plasma and widely used for therapeutic purpose. In this study, the mechanism of polymer formation and denaturing factors of albumin were investigated using purified human albumin. Moreover, many biochemical analyzes were performed to study the r...
Albumin is the protein of the highest concentration in plasma and widely used for therapeutic purpose. In this study, the mechanism of polymer formation and denaturing factors of albumin were investigated using purified human albumin. Moreover, many biochemical analyzes were performed to study the relationships between albumin and ceruloplasmin. The present studies also demonstrate and advanced purification method for ceruloplasmin from albumin. The human albumin was purified by cold ethanol precipitation and DEAE-Sepharose column chromatography. HPLC and counter-current immunoelectrophoresis analyze revealed that the purified human albumin also contained ceruloplasmin, haptoglobin, and α-1-lipoporein. When the human albumin incubated at 60℃ for l0hr in pH 7.0 conditions, the total lipid concentration was reduced and the solubility of albumin was dramatically increased compare than in pH 4.6 conditions. HPLC and FPLC analysis showed that the polymer form of albumin was increased after heat-treatment, whereas the dimer form of albumin was found predominantly before heat-treatment. In addition, the analysis of albumin by SDS-PAGE showed that the haptoglobin was not found both in pH 7.0 and pH 4.6 conditions after heat-treatment, whereas it found in both pH conditions before heat-treatment. This result suggests that haptoglobin is denatured during predenaturation of albumin by heat. On the other hand, the 0.22㎛ filtration could not remove efficiently the albumin dimer and polymer. During storage of the albumin for 2 years, the pH of albumin was slightly changed from 7.0 to 6.8 and the turbidity was not changed. However, the heme concentration in albumin was progressively increased with time in storage. Therefore, the color of albumin was changed after 1-year storage. It is suggested that the denaturation of albumin is not dependent in temperature but dependent increase in fatty acids during the storage. Albumin was more purified by DEAE-Sephacel and Prep-Cell column chromatography. Ceruloplasmin was purified by QAE-Sephadex A-50 and DEAE-Sepharose CL-6B column chromatography. Albumin and ceruloplasmin have oxidase activity. When stabilizer was added with heat-treatment, the oxidase activity of these proteins was decreased. This result indicates that stabilizer could inhibit the activity of ceruloplasmin. The ferroxidase activity was decreased and with increasing time in storage. Therefore, it is suggests that ceruloplasmin is binding protein with albumin in vivo and ceruloplasmin and albumin functions as anti-oxidative protein. By contrast with the oxidative activity, the peroxidase activity of albumin, which complemented with ceruloplasmin, was increased by heat-treatment and stabilizer. Furthermore, the ferroxidase activity of albumin, which completed with ceruloplasmin, was increase by heat-treatment, whereas the stabilizer inhibits ferroxidase activity. Therefore, temperature is an important factor for purifies ceruloplasmin from albumin.
Albumin is the protein of the highest concentration in plasma and widely used for therapeutic purpose. In this study, the mechanism of polymer formation and denaturing factors of albumin were investigated using purified human albumin. Moreover, many biochemical analyzes were performed to study the relationships between albumin and ceruloplasmin. The present studies also demonstrate and advanced purification method for ceruloplasmin from albumin. The human albumin was purified by cold ethanol precipitation and DEAE-Sepharose column chromatography. HPLC and counter-current immunoelectrophoresis analyze revealed that the purified human albumin also contained ceruloplasmin, haptoglobin, and α-1-lipoporein. When the human albumin incubated at 60℃ for l0hr in pH 7.0 conditions, the total lipid concentration was reduced and the solubility of albumin was dramatically increased compare than in pH 4.6 conditions. HPLC and FPLC analysis showed that the polymer form of albumin was increased after heat-treatment, whereas the dimer form of albumin was found predominantly before heat-treatment. In addition, the analysis of albumin by SDS-PAGE showed that the haptoglobin was not found both in pH 7.0 and pH 4.6 conditions after heat-treatment, whereas it found in both pH conditions before heat-treatment. This result suggests that haptoglobin is denatured during predenaturation of albumin by heat. On the other hand, the 0.22㎛ filtration could not remove efficiently the albumin dimer and polymer. During storage of the albumin for 2 years, the pH of albumin was slightly changed from 7.0 to 6.8 and the turbidity was not changed. However, the heme concentration in albumin was progressively increased with time in storage. Therefore, the color of albumin was changed after 1-year storage. It is suggested that the denaturation of albumin is not dependent in temperature but dependent increase in fatty acids during the storage. Albumin was more purified by DEAE-Sephacel and Prep-Cell column chromatography. Ceruloplasmin was purified by QAE-Sephadex A-50 and DEAE-Sepharose CL-6B column chromatography. Albumin and ceruloplasmin have oxidase activity. When stabilizer was added with heat-treatment, the oxidase activity of these proteins was decreased. This result indicates that stabilizer could inhibit the activity of ceruloplasmin. The ferroxidase activity was decreased and with increasing time in storage. Therefore, it is suggests that ceruloplasmin is binding protein with albumin in vivo and ceruloplasmin and albumin functions as anti-oxidative protein. By contrast with the oxidative activity, the peroxidase activity of albumin, which complemented with ceruloplasmin, was increased by heat-treatment and stabilizer. Furthermore, the ferroxidase activity of albumin, which completed with ceruloplasmin, was increase by heat-treatment, whereas the stabilizer inhibits ferroxidase activity. Therefore, temperature is an important factor for purifies ceruloplasmin from albumin.
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