Ribonucleases of human serum, urine, cerebrospinal fluid, and leukocytes. Activity staining following electrophoresis in sodium dodecyl sulfate-polyacrylamide gels.

Abstract

The ribonucleases (RNases) of human blood serum, urine, cerebrospinal fluid (CSF), and leukocytes were visualized by activity staining after electrophoresis in RNA-case sodium dodecyl sulfate-polyacrylamide gels. Samples were prepared for electrophoresis by heating for 2 min at 100 degrees C in 2% sodium dodecyl sulfate (NaDodSO4) and 5% mercaptoethanol, conditions which dissociate proteins into their constituent polypeptide chains and permit estimation of molecular weight. It was found that each of the five peaks of serum alkaline RNase activity separable on phosphocellulose columns, i.e., RNases 1-5 of Akagi et al. [Akagi, K., Murai, K., Hirao, N., & Yamanaka, M. (1976) Biochim. Biophys. Acta 442, 368-378], is associated with electrophoretically distinct enzymes. The molecular weights exhibited by these enzymes in NaDodSO4 gels are 31 000 and 28 000 (major species of RNase 1), 25 000 (RNase 2), 20 000 (RNase 3), 16 000 (RNase 4), and 14 000 (RNase 5). The RNase activity of leukocytes displays a molecular weight of 17 000 and exhibits a characteristic dependence of its Rf on the temperature at which samples (in 2% NaDodSO4 without mercaptoethanol) are prepared for electrophoresis. An RNase activity like that of leukocytes, distinct from RNases 1-5, is found in serum. Urine RNase activity is less heterogeneous than that of serum, consisting mainly of species like serum RNase 1 and an enzyme similar to leukocyte RNase. Conversely, CSF RNase activity is more complex and includes enzymes resembling serum RNases 1-5 as well as additional species either not observed in serum or detected in serum as minor components following chromatography. The analytical methods described herein are particularly useful for assessment of heterogeneity of RNase preparations and for direct comparison of the RNases of crude and purified samples.