Purification of α2-macroglobulin from Cohn Fraction IV by immobilized metal affinity chromatography: A promising method for the better utilization of plasma

• Published in: Journal of chromatography. B, Analytical technologies in the biomedical and life sciences Analytical technologies in the biomedical and life sciences, 2016-07, Vol.1025, p.68-75
• Main Authors: Huangfu, Chaoji, Ma, Yuyuan, Lv, Maomin, Jia, Junting, Zhao, Xiong, Zhang, Jingang
• Format: Article
• Language: English
• Subjects: alpha-Macroglobulins - analysis; alpha-Macroglobulins - chemistry; alpha-Macroglobulins - isolation & purification; Better utilization of plasma; Blood Proteins - chemistry; Chromatography, Affinity - methods; Cohn fraction IV; Humans; Hydrogen-Ion Concentration; Immobilized metal affinity chromatography; Methylamines; Plasma derivatives; Tandem Mass Spectrometry; Temperature; α2-macroglobulin
• ISSN: 1570-0232; 1873-376X
• DOI: 10.1016/j.jchromb.2016.05.013

•This is the first study that Cohn Fraction IV was used to purify α2-macroglobulin, which makes better use of human plasma.•Yield of 45% and purity of 95% were achieved simultaneously regardless of haptoglobin type.•Simple process of salting-out method and immobilized metal affinity chromatography facilitate pilot scale preparation.•α2-M could be readily inactivated on the condition of temperature above 50°C, pH above 9.0 or below 4.0, and the existence of methylamine.•This is the first study that Cohn Fraction IV was used to purify α2-macroglobulin, which makes better use of human plasma.•Yield of 45% and purity of 95% were achieved simultaneously regardless of haptoglobin type.•Simple process of salting-out method and immobilized metal affinity chromatography facilitate pilot scale preparation.•α2-M could be readily inactivated on the condition of temperature above 50°C, pH above 9.0 or below 4.0, and the existence of methylamine. As an abundant plasma protein, α2-macroglobulin (α2-M) participates widely in physiological and pathological activities including coagulation regulation, antitumor activities, and regulation of cytokines. It also presents a therapeutic potential for radiation injury. A two-step isolation method for the purification of α2-M from Cohn Fraction IV is described. This process includes a salting-out method and immobilized metal affinity chromatography. The LC-ESI–MS/MS analysis and a comparison of the amino acid composition demonstrated that the final product was α2-M. The final protein, with a purity of approximately 95% and a yield of nearly 45%, was obtained from Cohn Fraction IV regardless of plasma haptoglobin type, although all but type 1-1 have previously been considered unfavorable for α2-M preparation. The effects of temperature, pH, and methylamine on α2-M activity were evaluated to avoid activity loss during preparation and preservation. The results suggested that α2-M activity could be readily inactivated at temperatures above 50°C, at pH levels above 9.0 or below 4.0, or in the presence of methylamine. Cohn Fraction IV is usually discarded as a biological waste product in the human serum albumin production process; because the simple process developed in this study is relatively inexpensive, the preparation of α2-M from Cohn Fraction IV may better utilize human plasma, a valuable resource.