Methylamine Buffer Research Articles

327 Coamatic ® plasmin inhibitor: A new specific method for determination of plasmin inhibitor activity in plasma

It has recently been described that many commercial methods for the determination of plasmin inhibitor (PI) activity are not specific at low plasmin inhibitor concentrations (1, 2). This non-specificity results in values of 10-30 % when analysing PI-deficient plasmas. We have now developed a sensitive method which meets the criteria for a specific PI- activity method (3). Low plasmin concentrations (< 0.3 nkat/mL) and short incubation times (< 60 s) are used in combination with the highly selective chromogenic plasmin substrate, S-2403. With methylamine included in the buffer, no significant interference is observed when adding alpha-2-macroglobulin in excess (9 μM) to a Pi-deficient plasma. The specificity was further investigated and no influence from the non-plasminogen binding form of plasmin inhibitor (0.3 μM) or EACA (< 4 mM) was observed. The resulting kit contains; plasmin, plasmin solvent, methylamine buffer and S-2403. The kit has been adapted for the ACL 300R, Cobas Mira, MLA Electra 1000C and the Thrombolyzer instrument beside microplate and manual procedures. All the instrumentspecific methods results in an activity of < 6 % when analysing either a commercially Pi-deficient plasma or plasma from a congenitally Pi-deficient individual. Conclusion: The new Coamatic Plasmin Inhibitor kit provides instrument-specific methods that meet the criteria for valid methods for the determination of plasmin inhibitor activity.

Kinetic studies on the cleavage of phthalimide in methylamine buffers

In aqueous methylamine buffers of pH 10.21–11.25 nucleophilic cleavage of ionized (S−) phthalimide and general base-catalyzed cleavage of nonionized (SH) phthalimide is observed.

Chemical and enzymatic preparation of acylglycerols containing C18 furanoid fatty acids.

C18 furanoid triacylglycerol [glycerol tri-(9,12-epoxy-9,11-octadecadienoate)] was prepared by chemical transformation of triricinolein isolated from castor oil. The procedure involved oxidation, epoxidation and cyclization of the epoxy-keto intermediate with sodium azide and ammonium chloride in aqueous ethanol. The furanoid triacylglycerol was also obtained by esterification of C18 furanoid fatty acid with glycerol using Novozyme 435 (Novo Nordisk A.S., Bagsvaerd, Denmark) as biocatalyst. When Lipozyme (Novo Nordisk A.S.) was used, a mixture of the furanoid 1(3)-rac-monoacylglycerol and 1,3-diacylglycerol was obtained. In order to obtain the C18 furanoid 1,2(2,3)-diacylglycerol, selective hydrolysis of the furanoid triacylglycerol was achieved using porcine pancreatic lipase in tris(hydroxymethyl) methylamine buffer. Interesterification of triolein with methyl C18 furanoid ester in the presence of Lipozyme showed maximum incorporation of 34% of furanoid fatty acid. Extension of the interesterification to vegetable oils (olive, peanut, sunflower, corn and palm oil) allowed a maximum of 24% furanoid acid incorporation to be achieved.

Derivation of CO2 output from oscillations in arterial pH.

Theory predicts that the rate of rise of the oscillation in arterial CO2 partial pressure (PaCO2) is linearly dependent on CO2 flux from venous blood to alveolar gas. We have measured, in the anesthetized cat, CO2 output (VCO2) and oscillations in arterial pH. The pH signal was differentiated to give the maximum rate of fall of pH on the downstroke of the oscillation (dpH/dt decreases max). Since oscillations in pH are due to oscillations in arterial PCO2, dpH/dt decreases max was considered to be equivalent to the maximum rate of rise of the PCO2 oscillation. VCO2 was increased by ventilating the intestines with CO2 and by the intra-arterial infusion of 2,4-dinitrophenol. VCO2 was decreased by filling the intestines with isotonic tris(hydroxymethyl)methylamine buffer. The maximum range of VCO2 covered was 7.8-51 ml/min, and the mean range was from 13.6 +/- 1.3 to 29.7 +/- 1.6 (SE) ml/min. Although CO2 loading produced a small rise and CO2 unloading a small fall in mean PaCO2, the changes were not statistically significant, so that overall the response was close to isocapnia. Over the limited range of VCO2 studied there was a highly significant linear association between dpH/dt decreases max and VCO2 which supports the contention that the slope of the upstroke of the PaCO2 oscillation is determined by the CO2 flux from mixed venous blood to alveolar gas. As such this slope is a potential chemical signal linking ventilation to CO2 production.

Analysis of Guanase by Agarose Gel Electrophoresis and Activity Staining

A method was developed to separate guanase by agarose gel electrophoresis and to detect its activity by staining of the bands with a mixture of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NADP+, and a substrate of guanine, ethanol, phenazine methosulfate, nitrotetrazolium blue, and KCN in Tris-(hydroxymethyl)methylamine buffer (pH 8.0). Serum samples showed bands 1 (faster moving) and 2 corresponding to the positions of albumin and alpha 2-globulin, respectively, found by serum protein staining. The same bands were detected with guanase from human liver and kidney, although band 2 from the latter samples was not as distinct as that from the liver samples.

Quaternary structure of the pyruvate dehydrogenase multienzyme complex of Bacillus stearothermophilus studied by a new reversible cross-linking procedure with bis(imidoesters).

The bis(amidine) cross-links formed between protein subunits by treating them with bis(imidoesters) were found to be rapidly broken by exposing the cross-linked proteins to methylamine buffers containing the aprotic solvent acetonitrile. This cleavage step could be introduced between the two dimensions of a diagonal gel electrophoretic separation of cross-linked proteins to facilitate identification of the contributors to a cross-linked species. Tests with the tetrameric enzyme glyceraldehyde-3-phosphate dehydrogenase demonstrated the simplicity and effectiveness of the technique. When the pyruvate dehydrogenase multienzyme complex from Bacillus stearothermophilus was treated with a range of bis(imidoesters), from dimethyl succinimidate to dimethyl suberimidate, the most informative set of products was obtained with dimethyl glutarimidate. The longer bis(imidoesters) caused too extensive cross-linking of the enzyme subunits, although the beta chain of the pyruvate decarboxylase component always appeared to be the most resistant. Almost all the cross-linked species up to pentamers of the lipoate acetyltransferase polypeptide chain (apparent Mr approximately 280 000) were identified by means of the diagonal gel electrophoretic procedure after cleavage of the cross-links. The introduction of the methylamine cleavage step enables the bis(imidoester) for such experiments to be selected purely for the efficacity of its cross-linking reaction with the protein and dispenses with the need to incorporate a specially cleavable bond in the reagent.

Influence of erythrocyte iodothyronine-binding proteins on radioimmunoassay of thyroxin in dried blood spots.

Three erythrocyte proteins, one identified as hemoglobin, bind thyroid hormones. Using a dextran/charcoal radioimmunoassay for thyroxin in dried blood spots, we demonstrate that such binding differs with the buffer used. Barbital, phosphate, and borate buffers significantly enhance the binding more than glycine and tris(hydroxymethyl)methylamine buffers. Binding is not affected by agents commonly used to inhibit thyroxin binding to serum proteins. A highly significant nonlinear direct relationship between sample storage (temperature and duration) and increased thyroxin-erythrocyte binding is documented, together with an associated decrease in assayed concentrations of thyroxin. However, concomitant serial measurement of thyroxin with polyethylene glycol and combined double-antibody/polyethylene glycol radioimmunoassays produced no evidence of interference by erythrocyte proteins in the radioimmune reaction. We conclude that erythrocyte proteins act only as low-affinity secondary binders in radioimmunoassay for thyroxin.

Rapid removal of acetimidoyl groups from proteins and peptides. Applications to primary structure determination.

Methylamine buffers can be used for the rapid quantitative removal of acetimidoyl groups from proteins and peptides modified by treatment with ethyl or methyl acetimidate. The half-life for displacement of acetimidoyl groups from fully amidinated proteins incubated in 3.44 M-methylamine/HCl buffer at pH 11.5 and 25 degrees C was approx. 26 min; this half life is 29 times less than that observed in ammonia/HCl buffer under the same conditions of pH and amine concentration. Incubation of acetimidated proteins with methylamine for 4 h resulted in greater than 95% removal of acetimidoyl groups. No deleterious effects on primary structure were detected by amino acid analysis or by automated Edman degradation. Reversible amidination of lysine residues, in conjunction with tryptic digestion, has been successfully applied to the determination of the amino acid sequence of an acetimidated mouse immunoglobulin heavy chain peptide. The regeneration of amino groups in amidinated proteins and peptides by methylaminolysis makes amidination a valuable alternative to citraconoylation and maleoylation in structural studies.

Feasibility of measuring free and total bilirubin electrochemically in serum.

Bilirubin undergoes irreversible reduction at the hanging-mercury-drop electrode in tris(hydroxymethyl)methylamine buffer (0.1 mol/L, pH 8.5). Peak current is linearly related to concentration in the range 0-1 mg/L. The half-wave potential of -1.32 V vs the saturated calomel electrode is little affected by pH, whereas peak current increases with pH to pH 9.5, then decreases rapidly. Exposing the test solution to light causes the peak current to decline at a rate proportional to concentration. Albumin-bound bilirubin is electrochemically inactive, so free bilirubin can be measured in the presence of bilirubin-saturated albumin. Serum total bilirubin can be measured after much of the protein is precipitated by adding methanol. Results for serum bilirubin as measured electrochemically (y) in 20 methanolic filtrates of serum compared well with the Jendrassik-Grof Method (x) in preliminary studies: y = 0.981x -0.311; r = 0.9716. We discuss the possible mechanism of the reduction process and potential clinical applications. Polarographic measurement of bilirubin and bilirubin-binding capacity is feasible and may be advantageous as compared with existing techniques.

Kinetic measurement of guanine deaminase in serum with a centrifugal analyzer.

We describe an enzymic, one-step kinetic method for determination of guanine deaminase (guanase, EC 3.5.4.3) in serum with a centrifugal analyzer. A combined enzyme-substrate system consists of the enzymes xanthine oxidase, catalase, and aldehyde dehydrogenase, the coenzyme NAD+, the substrate guanine, and ethanol in tris(hydroxymethyl)methylamine buffer, with KCl added as activator for aldehyde dehydrogenase. The method requires only 40 microL of sample. Guanase activity in 28 samples can be determined within 10 min by setting a 4-min lag period. The increase in absorbance at 340 nm is linearly proportional to the activity of guanase to 60 U/L. Within-run precision (CV) was 1.32 to 4.50% over the range studied. Day-to-day precision corresponds to CVs of 4.8 to 7.2% over the same range of guanase activity. The reference interval, as calculated from data on 25 healthy humans, was 0 to 1.02 U/L. The enzymic automated method shows good correlation with Caraway's (Clin. Chem. 12: 187, 1966) method (r = 0.949).

Aspartate aminotransferase activity in human serum. Factors to be considered in supplementation with pyridoxal 5'-phosphate in vitro.

The pyridoxal phosphate reactivation of the apo form of aspartate aminotransferase (EC 2.6.1.1) in human serum has been studied with "normal" and above-normal activity of this enzyme. The extent of the reactionation did not depend on the presence of the substrates, L-aspartate or 2-oxoglutarate. Reactivation was greatest with 110 mumol of added pyridoxal phsophate present per liter during a preinucation for 7 min in tris(hydroxymethyl)methylamine buffer wit;h serum volume fractions ranging from 0.017 to 0.267. In comparison with measurements prformed with no exogenous pyridoxal phosphate present, we found two potential sources of error when this cofactor was added: (a) reagent and sample blanks in the pyridoxal phosphate-supplemented system were two- to eightfold higher and (b) progress curves were nonlinear when L-aspartate rather than 2-oxoglutarate was used as the startin substrate. Aspartate aminotransferase measurement sith pyridoxal phosphate supplementation was slightly more precise than without.

Enzymatic, fluorometric assay of alpha-amylase in serum.

An enzymatic, fluorometric method for kinetic assay of serum alpha-amylase (1,4alpha-D-glucan 4-glucanohydrolase, EC 3.2.1.1) is described. A soluble starch is used as substrate, in tris(hydroxymethyl)methylamine buffer. All measurements are made in Pyrex cuvettes at 37 degrees C, with a reaction volume of 1.16 ml. The assay is based on the following reaction sequence: (see article) The rate of appearance of fluorescence of NADH (lambdaex = 365 nm, lambdaem = 460 nm), developed in the indicator reaction (4), is measured and equated to the activity of alpha-amylase in serum. A calibration plot of the change of fluorescence per min vs. enzyme concentration shows a good proportionality in the range of 0.50-5.0 kU/liter.

Physicochemical Properties of the Hemoglobins from the Common Bloodworm Glycera dibranchiata

Abstract Glycera dibranchiata hemoglobin was previously noted to comprise five components separable by electrophoresis, two of which were due to monomeric forms and the remaining three due to polymeric forms. The three polymeric carboxyhemoglobin components had isoelectric points (pI) of pH 5.6, 6.1, and 6.5 while those of the monomeric proteins were pH 7.1 and 7.4. Oxidation of the pigment to methemoglobin caused an increase in each isoelectric point of 0.4 to 0.7. The spectral properties of the oxy-, deoxy-, carboxy-, met-, cyanomet-, and azide forms of the polymeric hemoglobin closely resembled those of vertebrate hemoglobins while analogous forms of the monomeric hemoglobin showed some anomalous properties, most notably in the high spin derivatives. The binding of hydroxide by methemoglobin at 4° indicated the formation of a normal hydroxide complex for the polymer (pK = 9.08, n = 0.98, where n represents the stoichiometry of the reaction) but a mixture of hydroxide and possibly imidazole methemoglobin complexes for the monomer. The pK for the transition in the monomer was 8.99 (n = 1.94) in the presence of methylamine buffer, while in glycine, pK = 10.08, n = 1.10. Studies of the binding of the monomeric methemoglobin with azide, cyanide, and fluoride at 22° gave the following dissociation constants. Kdissmethbcn = 1.2 x 10-3 m, n = 1.05, pH = 6.85 Kdissmethbn3 = 2.2 x 10-3 m, n = 0.97, pH = 6.45 Kdissmethbn3 = 2.7 x 10-3 m, n = 0.96, pH = 7.30 Kdissmethbf = 9.4 x 10-3 m, n = 1.01, pH = 6.85 where MetHbCn is cyanomethemoglobin, MetHbN3 is the azide form of methemoglobin, and MetHbF is fluoromethemoglobin. Amino acid analysis of preparative disc gel purified monomer indicated the presence of 144 amino acids yielding a globin molecular weight of ∼18,200 and a hemoglobin molecular weight of ∼18,800. The amino acid compositions of the nonpurified monomer and polymer were different from each other as well as from the purified monomer. The number of amino acids found in the nonpurified hemoglobin analyses was suggestive of the heterogeneity of these proteins.