Yphantis D A, Arakawa T
Department of Molecular and Cell Biology, University of Connecticut, Storrs 06268.
Biochemistry. 1987 Aug 25;26(17):5422-7. doi: 10.1021/bi00391a031.
Recombinant DNA derived human interferon gamma (IFN-gamma) from Escherichia coli was examined by equilibrium ultracentrifugation. Short-column equilibrium experiments at pH 6.9 in 0.1 M ammonium acetate buffer gave a z-average molecular weight of 33,500 +/- 1400 at infinite dilution, corresponding to 1.98 +/- 0.08 times the formula weight. Long- (2.6 mm) column experiments at pH 7.5 in 0.04 M imidazole buffer gave a molecular weight of 33,400 +/- 500. Under the latter conditions IFN-gamma behaves somewhat nonideally, with the departure from ideality accounted for by an effective (Donnan) charge of about 6+. No association of this dimer to form tetramer or higher polymers was observed, with the association constant for formation of tetramer from dimer K24 found to be less than 34 L mol-1. Similarly, no dissociation to monomers was observable, with the dissociation constant to monomer K21 being less than 5 X 10(-8) mol L-1. At pH 3.55 in 0.02 M buffer (acetate plus acetic acid), there was virtually complete dissociation of the dimer to monomer. Extreme nonideality was seen in this low ionic strength system, and the effective charge on the protein was estimated to be about 11+. The reduced molecular weight M(1 -upsilon rho) of the monomer was found to be about 4.09 +/- 0.20 kg mol-1; this corresponds to a molecular weight of 16,410 +/- 820, with the Scatchard definition of components. A small amount of a polymer with a molecular weight of about 0.5 X 10(6) was detected under these conditions.
通过平衡超速离心法对源自大肠杆菌的重组DNA衍生的人γ干扰素（IFN-γ）进行了检测。在pH 6.9的0.1 M醋酸铵缓冲液中进行的短柱平衡实验表明，在无限稀释时，z平均分子量为33,500±1400，相当于化学式量的1.98±0.08倍。在pH 7.5的0.04 M咪唑缓冲液中进行的长柱（2.6毫米）实验得出分子量为33,400±500。在后一种条件下，IFN-γ的行为有些非理想，偏离理想状态的原因是有效（唐南）电荷约为6+。未观察到该二聚体形成四聚体或更高聚合物的缔合现象，二聚体形成四聚体的缔合常数K24小于34 L·mol⁻¹。同样，也未观察到解离为单体的现象，解离为单体的解离常数K21小于5×10⁻⁸ mol·L⁻¹。在pH 3.55的0.02 M缓冲液（醋酸盐加醋酸）中，二聚体几乎完全解离为单体。在这种低离子强度系统中观察到了极端的非理想行为，估计蛋白质上的有效电荷约为11+。发现单体的折合分子量M(1-υρ)约为4.09±0.20 kg·mol⁻¹；根据斯查查德对组分的定义，这相当于分子量为16,410±820。在这些条件下检测到少量分子量约为0.5×10⁶的聚合物。
