Claudia M. Noyes

Comparison of five techniques for the determination of protein content in mixed human saliva

This study was conducted to assess the relative accuracy of five different assay techniques for the determination of protein concentration in human mixed saliva. The protein concentration of paraffin-stimulated saliva from 20 individuals was determined using the biuret reaction, the Lowry assay, a modified Lowry technique using bicinchoninic acid, and two dye-binding assays. Using bovine serum albumin as the standard, mean values ranged from 0.67 to 2.37 mg/ml. The use of bovine serum albumin, trypsinogen, lysozyme, bovine pancreatic ribonuclease, and poly-L-lysine as standards with the five different assay techniques to measure protein concentration of pooled mixed saliva from the above subjects produced results ranging from 0.74 to 65.5 mg/ml. The protein concentration obtained for this saliva sample by amino acid analysis was consistent with the value obtained for the biuret reaction using any of the five different standard proteins. Thus, the protein concentration obtained for human saliva depends upon both the technique used and the protein standard.

Optimization of complex separations in high-performance liquid chromatography : Application to phenylthiohydantoin amino acids

Abstract Graphical methods are described for determining suitable experimental conditions for optimum chromatographic resolution of complex mixtures. Plotting the logarithm of retention time for individual components or retention time ratios for close eluite pairs as a function of an experimental variable such as solvent strength or temperature allows direct visual evaluation of the best resolution attainable with a given column of known efficiency. Optimization of temperature and solvent strength for the separation of phenylthiohydantoin amino acids is demonstrated for two reversed-phase columns.

Cleavage and activation of human prothrombin by Echiscarinatus venom

The cleavage of human prothrombin by partially purified Echis carinatus venom (ECV) was investigated in the present report. Incubation of prothrombin with ECV resulted in the rapid cleavage of prothrombin to alpha-thrombin, with the release of fragment-1 and fragment-2. When dansyl arginine-N-(3-ethyl-1,5-pentanediyl) amide (DAPA), a very effective inhibitor of thrombin, was included in the ECV-prothrombin solution, meizothrombin was rapidly formed. Only small amounts of meizothrombin-1 could be detected. Prolonged incubation (23 h) in the presence of DAPA, however, resulted in nearly quantitative conversion of meizothrombin to meizothrombin-1 and fragment-1. Kinetic studies strongly suggested that the conversion of meizothrombin to meizothrombin-1 was due to ECV and not meizothrombin autolysis. In addition, EDTA, which inhibits ECV, blocked the cleavage of meizothrombin. Amino terminal sequence analysis indicated that ECV cleaves human prothrombin at two sites; Gly158-Ser159 and Arg322-Ile323. The former site differs from the site of autolytic cleavage of meizothrombin which occurs at Arg155-Ser156. In contrast to reports in the literature, the results of the present study indicate that the release of fragment-1 does not precede activation of human prothrombin by ECV.

Observations on the reaction of 2-hydroxy-5-nitrobenzyl bromide with a peptide-bound tryptophanyl residue

Previous studies on the isolation of peptides containing tryptophanyl residues modified with 2-hydroxy-5-nitrobenzyl bromide demonstrated multiple products of reaction at the same residue as well as technical difficulties in the primary structure analysis of peptides containing the modified tryptophanyl residue. The present study was undertaken to explore the reaction of 2-hydroxy-5-nitrobenzyl bromide with the single tryptophanyl residue in a synthetic peptide, experimental allergenic encephalitogenic peptide. The modification of this peptide was accomplished in sodium acetate, pH 4.75, and reagent removed by gel filtration. Amino acid analysis of the modified peptide suggested that only the tryptophanyl residue had been modified under these experimental conditions. The modified peptide could be separated into multiple derivatives by high-performance liquid chromatography. Although it is clear that some of the observed heterogeneity reflects a difference in the degree of substitution at the single tryptophanyl residue, several of the derivatives appear to have the same extent of substitution. It is suggested that the heterogeneity observed is a reflection of the establishment of a new diastereoisomeric center in the peptide. These results are consistent with previous observations from other laboratories and provide a basis for the explanation of apparent heterogeneity of peptides obtained from modified proteins.

Effect of divalent cations on the limited proteolysis of prothrombin by thrombin

The inhibitory influence of divalent cations on the ability of bovine alpha-thrombin to hydrolyze prothrombin showed the trend Mn2+ much greater than Ca2+ greater than or equal to Mg2+ greater than Sr2+ much greater than Ba2+. This effect was not due to an inhibition of thrombins catalytic activity as measured by hydrolysis of a specific synthetic substrate, H-D-Phe-pipecolyl-Arg-p-nitroanilide (D-PhePipArgNA). The presence of divalent cations did not inhibit thrombic proteolysis of gamma-carboxyglutamic acid (Gla)-domainless prothrombin. Prothrombin and Gla-domainless prothrombin were used as competitive inhibitors in the thrombic hydrolysis of D-PhePipArgNA. The apparent Ki value calculated for prothrombin was 18 microM. When either Ca2+ or Mn2+ were present, there was no inhibition. The apparent Ki value determined for Gla-domainless prothrombin was 28 microM in either the absence or presence of Ca2+. Addition of divalent cations to prothrombin, but not to Gla-domainless prothrombin, resulted in an altered protein conformation as measured by high-performance size-exclusion chromatography and ultraviolet difference spectroscopy. These results suggest that a conformational change secondary to the interaction of divalent cations with the Gla-containing domain of prothrombin is required for cation-dependent inhibition of thrombin hydrolysis.

Purification and Partial Characterization of Deoxyribonuclease I from Bovine Parotid Gland

Deoxyribonuclease I has been purified from bovine parotid gland. The purification procedure utilizes an acid extraction of minced parotid gland, salt fractionation, gel filtration, and ion-exchange chromatography. The last step, chromatography on Sulfopropyl-Sephadex, resolves the enzymatic activity into several fractions. The major fraction, designated DNase A, was subjected to further investigation. This enzyme has, as expected, an alkaline pH optimum and an obligate requirement for divalent cations. The presence of calcium chloride protects DNase A from inactivation by proteolytic enzymes. Despite the previously described immunologic dissimilarity, there appears to be a large amount of homology between the parotid and pancreatic DNases.

Activation of normal and abnormal human factor IX with trypsin.

Human factor IX is activated to factor IXa beta when factor XIa cleaves two peptide bonds, Arg 145-Ala 146 and Arg 180-Val 181, to release an activation peptide. In factor IX Chapel Hill (IXCH), isolated from a hemophilia B patient with a mild bleeding disorder, the arginine 145 residue has been replaced with a histidine. Thus factor IXCH is activated by factor XIa by cleaving only at the Arg 180-Val 181 bond, leaving the activation peptide attached, and resulting in an activated species, factor IXa alpha CH, that, like normal factor IXa alpha, is only 20% as active as factor IXa beta. It is reported that both factor IX and factor IXCH could be activated by trypsin to forms of factor IXa beta and factor IXa beta CH that had clotting activities identical to factor XIa-activated factor IX. Amino-terminal amino acid sequence analysis showed that trypsin cleaved factor IX at the same bonds as did factor XIa; factor IXCH was cleaved at the Arg 180-Val 181 bond, as normal, and was cleaved near the histidine 145, at the Lys 142-Leu 143 bond, releasing a slightly larger activation peptide than from normal factor IXa beta. Metal ions had no effect on the rate of activation of factor IX by trypsin; however, metal ions had a profound effect on the rate at which further incubation with trypsin inactivated factor IXa. Calcium and manganese protected factor IXa from inactivation by trypsin more effectively than magnesium, which was more effective than no metal ion. It is concluded that trypsin can activate normal factor IX and factor IXCH to fully active IXa beta forms.

Enzymatic inactivation of heparin cofactor II by a proteinase (proteinase-1) isolated from Echis carinatus venom

Heparin cofactor II was enzymatically inactivated by incubation with Echis carinatus venom in the presence of calcium. The initial rate of inactivation increased proportionately with the addition of heparin to a final concentration of 50 micrograms/ml. A proteinase, termed proteinase-1, was purified 17.5-fold from the venom which also enzymatically inactivated heparin cofactor II in the presence of calcium. The initial rate of heparin cofactor II inactivation by proteinase-1 was not increased by heparin at concentrations as high as 200 micrograms/ml. Heparin cofactor II was not inactivated by either unfractionated venom or proteinase-1 in the absence of calcium. The results indicate that heparin cofactor II, like antithrombin III, is susceptible to enzymatic inactivation by metalloproteinases in snake venoms.

Structural and Enzymatic Properties of Escherichia coli Glutamine Synthetase Subjected to Limited Proteolysis

Publisher Summary This chapter focuses on the structure of nicked glutamine synthetase. Glutamine synthetase is susceptible to limited proteolysis, termed nicking, under non-denaturing conditions by a variety of proteases. Proteolytic nicking occurs within a limited region of the enzyme and leads to a 97% reduction of enzymatic activity as measured by transferase and forward assays. Nicked glutamine synthetase retains its overall dodecameric structure, is still recognized by antibodies to intact enzyme, and is still adenylylated essentially normally. The chapter also describes the interactions of nicked glutamine synthetase with substrates and inhibitors using some experiments. The nicking of glutamine synthetase by protease under non-denaturing conditions does not completely inactivate the enzyme. Nicked glutamine synthetase catalyzes at least some of the same reactions as GS, but with a much lower efficiency than GS.