Summary. A colorimetric method for quantitative determination of noradrenaline is described. Determination of the oxidation rate at various pH for noradrenaline and adrenaline has shown that adrenochrome formation is complete after 1½ minutes at pH 4 whereas only some 10 p. c. of the maximal amount of noradrenochrome has been formed during the same time at pH 4. Noradrenochrome formation is complete in 2 minutes at pH 6. For routine determination adrenaline was oxidized at pH 4 for 1 1/2 minutes and noradrenaline at pH 6 for 3 minutes. Before removing excess iodine with thiosulphate the reaction may be adjusted to pH 4, where the bleaching effect of excess thiosulphate was minimal. A method for differential determination of adrenaline and noradrenaline depending on the different oxidation rates at pH 4 and 6 for the two substances is described. This method has been successfully applied to suprarenal extracts. The greater stability of noradrenaline in solution against oxidation as compared with adrenaline appears to be of practical value for example in solutions of local anaesthetics.
The trypsin binding was compared with preparations of a,-macroglobulin obtained from human plasma, serum or Cohn's fraction I11 by ammonium sulphate precipitation. Determination of the binding ratio a t various purification steps showed close to 1 : l molecular proportion, regardless of the degree of purity of the preparations. The a,-macroglobulin * trypsin complex was separated by gel chromatography on Sephadex G-200, using standardized conditions and estimating the bound and free trypsin in the eluate fractions. It was found that activated plasmin interfered with trypsin binding of a,-macroglobulin from Cohn's fraction 111.Immunologically pure a,-macroglobulin was obtained in six preparative steps from whole plasma after applying zone electrophoresis in polyvinylchloride. The protein was characterized by sedimentation rate, immunochemical analyses, isoelectric focusing, amino acid analysis and trypsin binding.Decreased binding capacity towards trypsin and plasmin could be detected after lyophylization of a,-macroglobulin. Comparison with low-temperature denaturation in sedimentation studies suggests that the functionally intact protein was not restored in spite of goo/, reaggregation of the 11-12-5 subunit. The structure dependence of the enzyme binding is discussed.The a,-macroglobulin is one of the major inhibitors of proteolytic activity in human plasma. The protein was isolated and characterized by Schultze et aZ. The earlier studies on complex formation with trypsin and some blood coagulation enzymes were done with partially purified a,-macroglobulin, serum or whole plasma. Recent purifications have aimed mainly a t preparing the native, immunologically pure protein . Such discrepancies and the fact that the trypsin or plasmin binding activity has not previously been studied in parallel with the purification of the human a,-macroglobulin [ 181 show that characterization of the functionally intact, purified protein is still incomplete. This report, dealing with the comparison of various methods to purify a,-macroglobulin provides information concerning the binding activity a t various stages of purification. Human whole plasma, serum or Cohn's fraction I11 precipitated with ammonium sulphate were used as starting materials. The amidase activity of the complex with trypsin was measured on N-benzoyl-DL-arginine p-nitroanilide [19,20], esterase activity on p-toluenesulphonyl-L-arginine methyl ester [8,9]. I n each case the complex was first separated by gel chromatography on Sephadex G-200. A decrease of the molecular binding activity was found with denatured a,-macroglobulin. The purified protein was characterized by sedimentation rate, immunological analyses and isoEur.
Pancreatic and urinary kallikreins failed to form the typical serine proteinase complex with alpha2M (alpha2-macroglobulin). Studies were performed to compare this with the binding of trypsin to alpha2M at various molar binding ratios, with the use of Sephadex G-200 gel filtration to separate free and alpha2M-bound enzyme fractions. The subunit conversion was totally absent with pancreatic kallikrein from lhich traces of a binding proteinase had been removed. The lack of binding is believed to be the result of the restricted specificity of the kallikreins.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.