TY - JOUR
T1 - Structural studies of apolipoprotein B
T2 - physical properties of the protein in guanidine hydrochloride
AU - Patterson, Bruce W.
AU - Miller, Nancy H.
AU - Fisher, Waldo R.
PY - 1987/8/15
Y1 - 1987/8/15
N2 - Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (ifMz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540000 ± 110000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25°C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640 000 and 250 000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.
AB - Apolipoprotein B was isolated from human plasma low-density-lipoprotein without precipitation by diethyl ether/ethanol extraction of the protein in 6 M guanidine hydrochloride. The physical properties of this protein, which contained a residuum of approximately 7% phospholipid, were examined in 6 M guanidine solution under reducing conditions. The circular dichroism spectrum was indistinguishable from that of a random coil protein. Sedimentation equilibrium analyses of apolipoprotein B by the meniscus depletion method of Yphantis (1984, Biochemistry 3, 297-317) were complicated by heterogeneity and nonideality despite the low concentrations employed. 63 analyses of the weight average (Mw) and z average (Mz) molecular weight were made on the apolipoprotein B from 12 subjects. The Mw observed was a function of initial concentration, rotor speed, and a heterogeneity index (ifMz/Mw). Multiple linear regression of apolipoprotein B molecular mass against these parameters suggested that an Mw of 540000 ± 110000 would be observed under apparently ideal and homogeneous conditions. The sedimentation coefficient and intrinsic viscosity of the reduced protein at 25°C in 6 M guanidine were 2.13 S and 116 ml/g, respectively; these values predict molecular weights of 640 000 and 250 000, respectively, if apolipoprotein B was fully denatured into a random coil. Lack of agreement between these estimates and with the sedimentation equilibrium analysis can best be explained by compactness of structure and incomplete denaturation to a random coil state. Furthermore, an irreversible temperature dependence of apolipoprotein B reduced viscosity indicated that residual structure remained in solutions of 6 M guanidine hydrochloride/20 mM dithiothreitol. Taken together, the physical data demonstrate that apolipoprotein is a single polypeptide of approximately 540 kDa, whose structure resists denaturation under conditions where most proteins exist as random coils.
KW - Apolipoprotein B
KW - Apolipoprotein structure
KW - Guanidine hydrochloride
KW - LDL
KW - Protein denaturation
UR - http://www.scopus.com/inward/record.url?scp=0023276312&partnerID=8YFLogxK
U2 - 10.1016/0005-2760(87)90104-4
DO - 10.1016/0005-2760(87)90104-4
M3 - Article
C2 - 3607081
AN - SCOPUS:0023276312
SN - 0005-2760
VL - 920
SP - 266
EP - 276
JO - Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
JF - Biochimica et Biophysica Acta (BBA)/Lipids and Lipid Metabolism
IS - 3
ER -