A haptoglobin radioassay based on binding to solid-phase hemoglobin.
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Animals↗
Biomedical subjects
A C Peacock
Publications and source records attributed to A C Peacock.
Conformational states of 18 S rRNA from HeLa cells detected by gel electrophoresis.
Gel electrophoretic analysis has been used to study some of the conformational properties of 18 S rRNA from HeLa cells. After isolation and treatment by heat or denaturants, the mobility of the RNA in 3% acrylamide/0.5% agarose composite gels was found to be altered; often there were two or more distinct components. Several of these forms could be interconverted by heating to 40 degrees C or by use of dimethyl sulfoxide. Although these comformers were easily distinguishable (by their electrophoretic properties), their melting profiles were very similar. Only a single electrophoretic form was detected when electrophoresis was performed at 40 degrees C. The conformational differences observed are probably derived by variations in base pairing and stacking that result when the parent form is cooled.
Electrophoresis, Polyacrylamide Gel↗
Bioassay for prolactin: densitometric analysis on polyacrylamide gels of milk protein production by mammary explants in vitro.
Mammary explants from mice in midpregnancy cultured in a synthetic medium containing insulin and hydrocortisone respond to the addition of prolactin by the synthesis of milk proteins. The secretory response in alveolar lumina has served as a histologic endpoint in a sensitive bioassay for prolactin developed by Kleinberg and Frantz (J Clin Invest 50: 1557, 1971). This report demonstrates that the quantitative densitometric analysis of stained milk proteins (caseins) made in response to prolactin is a useful modification of that bioassay. Electrophoretic analysis of the proteins extracted from the explants permits a quantitative estimate of casein content. The amount of casein present after 5 days of incubation was found to be a measure of the prolactin concentration in the medium. No radioactive isotopes are used. The use of electrophoretic analysis has practical advantages over the histologic scoring used earlier, and has approximately the same range of sensitivity.
Animals↗
Determination of the subunit composition of haptoglobin 2-1 polymers using quantitative densitometry of polyacrylamide gels.
Human haptoglobin (Hp), a hemoglobin-binding glycoprotein containing two types of polypeptide chains, alpha and beta, in equimolar amounts linked by disulfide bonds, exists in three major phenotypes determined by the properties of the alpha chain: Hp 1-1 (alpha1), Hp 2-2 (alpha2), and Hp 2-1 (alpha1 and alpha2). Hp 2-2 and Hp 2-1 form a series of alpha-disulfide-linked polymers. The subunit composition of the Hp 2-1 series was studied by isolation of single Hp 2-1 polymers by polyacrylamide gel electrophoresis. After reductive disulfide cleavage and alkylation the relative content of alpha2 and alpha1 polypeptide chains was determined by quantitative densitometry of acid/urea polyacrylamide gels stained with Coomassie brilliant blue R250. The molar ratios alpha2/alpha1 for the Hp 2-1 polymers. P1 through P5 (in order of decreasing electrophoretic mobility), were found to be: P1, 0.0 (alpha1 only); P2, 0.48; P3, 0.97; P4, 1.6; P5, 2.0. Since one alphabeta-Hb half-molecule is known to bind to each Hp beta chain, the beta polypeptide chain content of each of the Hp 2-1 polymers could be estimated by by counting the number of Coomassie blue bands formed after electrophoresis of isolated Hp 2-1 polymers fractionally saturated with cyanmethemoglobin (Hb). The number of beta chains present in Hp 2-1 polymers P1 through P4 was determined to be: P1, 2; P2, 3; P3, 4 and P4, 5. Molecular weights of the Hp 2-1 polymers were determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresus using as standards the almost homologous Hp 2-2 polymer series whose molecular weights are known from ultracentrifugation studies. Molecular weights for the first five Hp 2-1 polymers were estimated to be 107,000; 162,000; 217,000, 274,000; and 331,000, respectively. These data are consistent with the previously proposed model for the subunit composition of the Hp 2-1 polymer series when P1 = (alpha1 beta)2 and the subsequent polymers in order are represented as (alpha1 beta)2(alpha2 beta)n where n = 1,2,3,4...
Densitometry↗
Hemoglobin binding by isolated polymeric proteins from human haptoglobin types 2-1 and 2-2. Some suggested polymer subunit compositions.
1. Some of the individual members of the polymeric series of proteins from human haptoglobin types 2-1 and 2-2 were isolated by gel electrophoresis. By reacting this purified material with less than an equivalent amount of hemoglobin and analyzing the result by electrophoresis, the number of haptoglobin-hemoglobin complexes could be clearly counted. For the haptoglobin 2-1 series, the number of complexes formed was n+1, where n is the serial order, in decreasing electrophoretic mobility, of the haptoglobin polymeric form used. For the haptoglobin 2-2 series, the number of complexes was n+2. 2. For the first three members of haptoglobin 2-1 series, the haptoglobin-hemoglobin composition of the complexes was estimated from scans of the unstained gels. The data indicated that this series consists of 2,3,4... alpha beta haptoglobin subunits, each of which can combine with an alpha beta subunit of hemoglobin.
Electrophoresis, Polyacrylamide Gel↗