Physical properties of collagen-sodium dodecyl sulfate complexes

Abstract

Sodium dodecyl sulfate (NaDodSO4)--polyacrylamide gel electrophoresis and gel filtration chromatography of protein--NaDodSO4 complexes are frequently used to characterize collagen-like polypeptide components in mixtures obtained from extracts of basement membranes. However, electrophoresis yields anomalously high apparent molecular weights for collagenous polypeptides when typical globular proteins are used as molecular weight standards, and the use of gel filtration chromatography for this purpose was suspect because Nozaki et al. [Nozaki, Y., Schechter, N. M., Reynolds, J. A., & Tanford, C. (1976) Biochemistry 15, 3884--3890] found that asymmetric particles, including NaDodSO4--protein complexes, coeluted with native globular proteins of lower Stokes radius, when Sepharose 4B was used. To understand these effects and to improve the characterization of collagenous polypeptides, we investigated the secondary structure of NaDodSO4--collagen complexes with the use of circular dichroism, measured the NaDodSO4 content, studied the dependence of electrophoretic mobility on gel concentration, and extended work on gel filtration by use of a more porous gel, Sepharose CL-4B. We found that the anomalous behavior of collagen chains on NaDodSO4--polyacrylamide gel electrophoresis is due in large part to treatment of data and that the method can be used to determine rather accurate values for the number of residues per polypeptide chain. Our gel filtration results indicated that reliable molecular weights can be obtained when Sepharose CL-4B is used. These methods can be applied equally well to collagenous and noncollagenous polypeptides.

Amino acid sequences of hordein polypeptides

“…This implies that the electrophoresis technique overestimated the molecular weights of this hordein polypeptide, as has been observed for instance with collagen, which like the hordeins has a high proline content (13). In the ultracentrifuge studies of QUENSEL and SVEDBERG (20) from 1938 the hordein polypeptides sedimented in 65 % alcohol as one narrow, symmetrical peak with a sedimentation coefficient of 2.0, which was interpreted to signify a molecular weight of 27.500.…”

A procaryotic regulatory factor with a histone H1-like carboxy-terminal domain: clonal variation of repeats within algP, a gene involved in regulation of mucoidy in Pseudomonas aeruginosa

“…The observed size of the [3H]prolinelabeled polypeptide (48.6 kDa) was 40% higher than that of the algP gene product (AlgP) predicted from the DNA sequence (34,560 Da). This is, without exception, typical of proteins with repeated motifs rich in proline (22,23,67). In addition, AlgP has a high positive net charge (predicted pl 10.4), which may also contribute to the slower electrophoretic mobility observed on SDS-polyacrylamide gel electrophoresis.…”

Immunocytochemical analysis of AlgP (Hp1), a histonelike element participating in control of mucoidy in Pseudomonas aeruginosa

“…Although the observed molecular mass for the upper band exceeds the mass predicted from the algP sequence (34,560 Da), this has been attributed to the high proline content of AIgP (almost 25% for its carboxy-terminal half). Proline-rich proteins invariably display anomalous electrophoretic mobility in SDS-polyacrylamide gels (in the range of 140%o of the expected value when the proline content is as high as that in AlgP [17,18,38]). These experiments established that the polypeptides recognized by rabbit polyclonal antibodies were identical to those detected with monoclonal antibodies.…”