Characterization of monoclonal antibody to DNA.RNA and its application to immunodetection of hybrids.

Monoclonal antibodies were raised to a DNA.RNA heteropolymer duplex prepared by transcription of phi X174 single-stranded DNA with DNA-dependent RNA polymerase. A monoclonal antibody with the highest affinity and specificity was selected. This antibody bound the DNA.RNA heteropolymer and poly(I).poly(dC) equally but 100-fold higher levels of poly(A).poly(dT) were required to achieve a similar degree of binding in competitive binding assays using DNA.[3H]RNA. Single-stranded DNA, double-stranded DNA and RNA, and ribosomal RNA were not bound by the antibody. The observed association constant for the antibody and DNA.[3H]RNA, determined by Scatchard analysis, was 8.5 X 10(10) l/mol assuming independent antibody binding sites. The antibody and an alkaline phosphatase-labeled second antibody were used in an immunodetection method for measurement of hybrids formed between immobilized DNA probes of various lengths and 23 S ribosomal RNA. The colorimetric response of this assay increased linearly with the amount of hybrid formed.

Steroid-protein interactions. Human corticosteroid-binding globulin: characterization of dimer and electrophoretic variants.

Human corticosteroid-binding globulin (CBG) forms a dimer that was isolated by gel filtration, has full binding affinity and capacity, and can be dissociated to the monomer. Monomeric CBG consists of two distinct molecular variants, which were detected by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The two monomeric CBG species were separated by preparative gel electrophoresis and were found to bind cortisol, as well as progesterone, with equal affinity. They have one steroid binding site per CBG molecule. Amino acid and carbohydrate analyses are essentially the same for both of the CBG variants. Removal of sialic acid or 90% of the carbohydrate did not affect the existence of the two molecular forms. The two CBG species were isolated from each of the sera from five individual donors, indicating that the observed heterogeneity does not result from pooling genetic variants. The two species are immunologically identical. A possible explanation for the existence of the two electrophoretic variants is a difference in amidation.

Steroid-protein interactions. Human corticosteroid binding globulin: some physicochemical properties and binding specificity.

Reducing agents (dithiothreitol and beta-mercaptoethanol) significantly decrease the affinity constants of the human corticosteroid-binding globulin (CBG)-cortisol complex in proportion to their concentration; the resulting Ka values are more consistent than those obtained in the absence of the reductants. The effect is reversible. The equilibrium association constants of the CBG complexes with cortisol and progesterone show a relatively broad pH maximum between pH 8 and 11. In this pH range, cortisol was found to be bound more strongly than progesterone; this relationship is reversed around pH 6. The van't Hoff plot of the temperature effect on Ka of the CBG-cortisol complex (4-41 degrees C) exhibits a nonlinear, possibly biphasic temperature dependency. The shape of the van't Hoff plot was similar in the presence of mercaptoethanol. The association of cortisol and progesterone to human CBG at 4 and 37 degrees C is enthalpy driven, compensating for the unfavorable change in entropy. Studies with 47 steroids served to elucidate the influence on binding affinity of polar and nonpolar groups and other structural alterations. The contribution of specific structural changes in the steroid molecule to the free energy of binding can be calculated from the results. Important structures for optimal binding are the 20-oxo group, a 10 beta-methyl group, and a double bond at the 4 position. A complementary image of the binding site with respect to the nature of binding at various locations is proposed.

Purification and characterization of the corticosteroid-binding globulin of pregnant guinea pig serum.

The corticosteroid-binding globulin from guinea pig pregnancy serum was purified by the sequential use of affinity chromatography, hydroxylapatite chromatography, and gel filtration chromatography at a cumulative yield of 80%. The protein was found to be homogeneous by analytical gel electrophoresis, equilibrium sedimentation ultracentrifugation, immunoelectrophoresis, and stoichiometry (1:1) of steroid binding. Guinea pig corticosteroid-binding globulin has a molecular weight of 43 300 and contains 29% carbohydrate. The intrinsic fluorescence of the corticosteroid-binding globulin is quenched by about 73% when 1 mol of cortisol is bound. The association constants (pH 7.4) at 4 and 37 degrees C are 2.5 X 10(7) and 1.5 X 10(6) M-1 for cortisol and 1.4 X 10(6) and 0.2 X 10(6) M-1 for progesterone, respectively.

Purification and characterization of the sex steroid-binding protein of rabbit serum. Comparison with the human protein.

The sex steroid-binding protein (rSBP) of immature rabbit serum was purified to homogeneity by the sequential use of DEAE-cellulose chromatography, affinity chromatography on 5alpha-dihydrotestosterone-17 beta-succinyl-diaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, agarose (Bio-Gel-A-0.5m) gel filtration, and preparative polyacrylamide gel electrophoresis. The cumulative yield is 13%. Homogeneity of rSBP was shown by the equilibrium sedimentation ultracentrifugation in 6 M guanidine HCl containing 0.1 M mercaptoethanol which yields an average molecular weight of 36,475 +/- 865. Analytical gel electrophoresis in the presence of sodium dodecyl sulfate and gel filtration on agarose yield a molecular weight of 57,000 and 120,000, respectively. The variation is due to a 30% carbohydrate content. The amino acid composition is reported. Comparison of the rabbit and human SBP indicate that they are different in both their molecular and functional properties.

Characterization of the sex steroid binding protein of human pregnancy serum. Improvements in the purification procedure.

The sex steroid binding protein (SBP) of human pregnancy serum was purified to homogeneity by the sequential use of ammonium sulfate precipitation, affinity chromatography on 5alpha-dihydrotestosterone-17beta-succinyldiaminoethyl-(1,4-butanediol diglycidyl ether)-agarose, and preparative polyacrylamide gel electrophoresis. The yield of pure SBP was improved from 5% as originally reported [Mickelson, K. E., and Petra, P. H. (1975), Biochemistry 14, 957] to 34%. Homogeneity of SBP was shown by equilibrium sedimentation ultracentrifugation in 6 M guanidine hydrochloride containing 0.1 M mercaptoethanol which yields a minimum molecular weight of 36 335 +/- 525. The protein is also homogeneous when examined by gel electrophoresis in the presence of sodium dodecyl sulfate. A value of 52 000 for the molecular weight is obtained by this method. SBP partially purified from Cohn fraction IV has also a molecular weight of 52 000 by gel electrophoresis in the presence of sodium dodecyl sulfate; that fraction is contaminated with another protein of molecular weight 90 000 which must be removed to obtain homogeneous SBP. The amino acid composition of SBP isolated from pregnancy serum is presented.

Purification of the sex steroid binding protein from human serum.

The sex steroid binding protein from human pregnancy serum was purified to homogeneity by affinity chromatography and preparative polyacrylamide gel electrophoresis. The selective adsorbants were prepared by coupling [3H]-5alpha-dihydrotestosterone 17beta-hemisuccinate to 3,3'-diaminodipropylamine-agarose, poly(Lys-DLAla)-agarose, and albumin-agarose. The most effective adsorbant purifying for the binding protein was 5alpha-dihydrotestosterone 17beta-hemisuccinyl-3,3'-diaminodipropylamine-agarose. A preparative procedure with 5alpha-dihydrotestosterone 17beta-hemisuccinyl-3,3'-diaminodipropylamine-agarose yielded active material which was further purified by preparative polyacrylamide electrophoresis at pH 9.5. Homogeneity was shown by analytical disc gel electrophoresis at three different pH units. A single radioactive band corresponding to the stained band was shown by incubating with [1,2-3H]-5alpha-dihydrotestosterone prior to electrophoresis. The radioactive peak corresponding to the pure sex steroid binding protein could not be detected when a 100-fold excess of 17beta-estradiol was present in the incubation prior to electrophoresis demonstrating the specific sex steroid binding properties of this protein. The migration of this peak was identical with that obtained when diluted serum was electrophoresed under the same conditions in the presence of [1,2-3H]-5alpha-dihydrotestosterone indicating that no significant changes in the molecular characteristics of the binding protein occurred during the purification procedure. The presence of carbohydrate in the pure protein was shown by the periodic acid-Schiff reagent procedure. Selective adsorbants containing 17beta-estradiol linked at the 3 position were ineffective in retaining sex steroid binding protein activity.