An improved radioreceptor assay for 1,25-dihydroxyvitamin D in human plasma

We describe a modified assay technique for quantitating 1,25-dihydroxyvitamin D in plasma. The method involves a rapid extraction of the hormone using minicolumn (made of granular diatomaceous earth) chromatography followed by single-step purification on high-performance liquid chromatography. Quantitation of plasma 1,25-dihydroxyvitamin D is achieved by a radioligand receptor assay employing lyophilized cytosolic receptor protein from chick intestine and high-specific-activity 1,25-dihydroxy[³H]vitamin D₃ (166 Ci/mmol). A new incubation medium including an ethanol extract of vitamin D-deficient chick serum yields high specific binding and improves the precision of the radioassay. Bound and free hormone are separated with dextran-coated charcoal of equivalent particle size. The method is sensitive to 0.5 pg/tube with a practical detection range of 1–20 pg/tube, permitting duplicate assay of endogenous 1,25-dihydroxyvitamin D in plasma volumes as small as 0.5 ml. The intra- and interassay coefficient of variation are 5 and 9%, respectively, and the method is valid over a wide-range sample dilution. This assay technique was applied to the measurement of plasma 1,25-dihydroxyvitamin D hormone concentration in normal young adults (55.2 ± 13.6 pg/ml; n = 20) and in patients with chronic renal failure (13.5 ± 5.2 pg/ml; n = 9) and primary hyperparathyroidism (83.3 ± 18 pg/ml; n = 10).     

An improved spectrophotometric assay for superoxide dismutase based on epinephrine autoxidation

Superoxide dismutase activity was assayed in terms of its ability to inhibit the radical-mediated chain-propagating autoxidation of epinephrine. The enzyme assay based on adrenochrome absorption at 480 nm has been improved by measuring the absorption change at 320 nm. This alternative procedure was found to be 6 to 10 times more sensitive and more consistent than that measured at 480 nm.     

An improved spectrophotometric assay for prostaglandin synthetase based on the oxidation of epinephrine

The spectrophotometric assay method for prostaglandin synthetase from Takeguchi and Sih (1) was improved by monitoring the absorption change at 320 nm instead of at 480 nm during the enzymatic synthesis. The measurement at 320 nm is more sensitive and more consistent than the A₄₈₀ measurement. The improvement resulting from the measurement at 320 nm is attributed to a combination of factors, including a higher extinction coefficient, a more inclusive measurement of other epinephrine oxidative product(s) and lower interference due to the product of the further oxidation of adrenochrome. The validity of this spectrophotometric method was also verified in this report.     

An o-phthalaldehyde spectrophotometric assay for proteinases

A rapid and convenient spectrophotometric assay has been devised to measure proteolysis. The assay is based on the reaction of o-phthalaldehyde (OPA) and 2-mercaptoethanol with amino groups released during proteolysis of a protein substrate. The reaction is specific for primary amines in amino acids, peptides, and proteins, approaches completion within 1 to 2 min at 25 degrees C (half-times of approx 10-15 s), and requires no preliminary heating or separation of the hydrolyzed products from the undegraded protein substrate prior to performing the assay. The OPA assay was relatively as successful as a 2,4,6-trinitrobenzenesulfonic acid (TNBS) procedure in predicting the extent of hydrolysis of a protein substrate. The utility of the OPA method was demonstrated by measuring the degree of proteolytic degradation caused by trypsin, subtilisin, Pronase, and chymotrypsin of various soluble protein substrates. Ethanethiol (instead of 2-mercaptoethanol) or 50% of dimethyl sulfoxide can be included in the assay solution to stabilize certain OPA-amine products. The present method approaches the sensitivity of ninhydrin and TNBS procedures, is more convenient and rapid, and could substitute for these reagents in most assay systems.     

Serum carboxypeptidase B: A spectrophotometric assay using protamine as substrate

A quantitative colorimetric assay for serum carboxypeptidase B (SCPB, anaphylatoxin inactivator, kininase I) is described. SCPB is known to possess an enzymatic specificity for cleaving COOH-terminallysyl and arginyl residues which is similar to the specificity of bovine pancreatic carboxypeptidase B. One function of SCPB involves the inactivation of C3a and C5a, the two complement derived anaphylatoxins. Since cobalt markedly enhances the activity of the enzyme, serum is treated with CoCl₂ before the SCPB assay is performed. Salmine, a protamine from salmon sperm, was selected as the substrate because it contains multiple COOH-terminal arginyl residues and is digested more rapidly by SCPB than other common substrates of carboxypeptidase B, including hippuryl-arginine and benzyl-glycylarginine. The kinetics for arginine release from salmine were first-order throughout the course of the assay and the colorimetric values obtained were related to micromols of arginine released. A unit of SCPB is defined as one nanomol of arginine released per minute per milliliter of serum. The range of SCPB activity in serum from healthy individuals was found to be 318 to 466 units. The medians of SCPB activity in sera obtained from patients with Dengue shock syndrome and with shock following intravenous dextran infusion were both lower than the mean SCPB activity of healthy individuals. SCPB levels in patients homozygous and heterozygous for cystic fibrosis were within the normal range.     

An ultraviolet spectrophotometric assay for aspartate transcarbamylase

A procedure for determining the activity of aspartate transcarbamylase, based upon the greater ultraviolet absorbancy of the products of the reaction catalyzed compared to the reactants, was devised. Extinction coefficients were determined at 205, 210, and 215 nm for the compounds carbamoyl aspartate, acetyl aspartate, and aspartate. These values formed the quantitative basis for a spectrophotometric assay in which an enzymatic reaction is monitored at one of these wavelengths. Use of this procedure was illustrated in four kinetic experiments with the allosteric aspartate transcarbamylase from Escherichia coli, and the nonallosteric catalytic subunit of this enzyme: aspartate saturation curve, arsenate saturation curve (reverse reaction), allosteric activation by a transition-state analog employing acetyl phosphate as substrate, and carbamoyl phosphate progress curve (substrate depletion in the presence of excess cosubstrate). Owing to changes in absorbance on the order of 1000 liter mol^?1 cm^?1 concomitant with the reaction, the sensitivity of the method is comparable to that of many procedures already in the literature.     

An improved radioenzymatic assay for plasma norepinephrine using purified phenylethanolamine N-methyltransferase

Radioenzymatic assays have been developed for norepinephrine (NE) using either catechol O-methyltransferase (COMT) or phenylethanolamine N-methyltransferase (PNMT). Assays using PNMT are specific for NE but have been considered less sensitive than the more complex assay procedures employing COMT. An improved purification procedure for bovine PNMT has permitted development of a NE assay with substantially improved sensitivity (less than 0.5 pg), reproducibility, and decreased manipulative effort. PNMT was purified by sequential pH 5.0 treatment and dialysis and by column chromatographic procedures using DEAE-Sephacel, Sephacryl S-200 and Phenyl Boronate-agarose. Recovery of PNMT activity through the purification scheme was 50% while blank recovery was less than 0.001%. Norepinephrine can be directly quantified in 25 microliters of human plasma and a seventy-tube assay can be routinely completed within 4 h. The capillary to venous plasma NE gradient was examined in eight normotensive male subjects. Capillary plasma NE (211 +/- 21.7 pg/ml) was significantly lower than venous plasma NE (367 +/- 32.7 pg/ml) in all subjects (p less than 0.005). This difference suggests the concentration of NE in capillary blood may be a unique indicator of sympathetic nervous system activity in vivo.     

An improved dispersed adrenal cell assay for corticotropin in rat plasma

The present study was designed to improve the dispersed adrenal cell technique for determining adrenocorticotrophic hormone (ACTH) concentrations in small amounts of rat plasma. Priming with ACTH, incubation with methyl-isobutylxanthine, or dexamethasone pre-treatment were employed as modifications. Of these, only dexamethasone pre-treatment increased the sensitivity of the assay. The adrenal fragments obtained from 10-12 adult male rats pre-treated with dexamethasone (100 micrograms/kg B.W.) one hour before sacrifice, were digested with collagenase and deoxyribonuclease solution for 30 minutes. The dispersed cells were collected by centrifugation and resuspended in Krebs-Ringer bicarbonate buffer containing 0.2% glucose and 0.5% bovine serum albumin. Aliquots of cell suspension (3-4 X 10(4)/tube) were incubated with various doses of ACTH1-24 or the eluate of plasma samples at 37 degrees C for 2 hours in an atmosphere of 95% O2/5% CO2 in a Dubnoff shaker. The quantity of corticosterone produced was measured fluorimetrically. The assay is precise (lambda = 0.06), extremely sensitive (10 fg/tube), and convenient. One skilled technician can handle 15 to 20 plasma samples per day using 10 rats as the source of assay cells. ACTH can be measured in as little as 10-50 microliters of eluate.     

Inactivation of bradykinin by angiotensin-converting enzyme and by carboxypeptidase N in human plasma

Because bradykinin (BK) appears to have cardioprotective effects ranging from improved hemodynamics to antiproliferative effects, inhibition of BK-degrading enzymes should potentiate such actions. The purpose of this study was to find out which enzymes are responsible for the degradation of BK in human plasma. Human plasma from healthy donors (n = 10) was incubated with BK in the presence or absence of specific enzyme inhibitors. At high (micromolar) concentrations, BK was mostly (>90%) degraded by carboxypeptidase N (CPN)-like activity. In contrast, at low (nanomolar) substrate concentrations, at which the velocity of the catalytic reaction is equivalent to that under physiological conditions, BK was mostly (>90%) converted into an inactive metabolite, BK-(1-7), by angiotensin-converting enzyme (ACE). BK-(1-7) was further converted by ACE into BK-(1-5), with accumulation of this active peptide. A minor fraction (<10%) of the BK was converted into another active metabolite, BK-(1-8), by CPN-like activity. The present study shows that the most critical step in plasma kinin metabolism, i.e., inactivation of BK, is mediated by ACE. Thus inhibition of plasma ACE activity would be cardioprotective by elevating the concentration of BK in the circulation.     

An enzyme-coupled continuous spectrophotometric assay for glycogen synthases

The metabolic pathways leading to the synthesis of bacterial glycogen involve the action of several enzymes, among which glycogen synthase (GS) catalyzes the elongation of the α-1,4-glucan. GS from Agrobacterium tumefaciens uses preferentially ADPGlc, although UDPGlc can also be used as glycosyl donor with less efficiency. We present here a continuous spectrophotometric assay for the determination of GS activity using ADP- or UDPGlc. When ADPGlc was used as the substrate, the production of ADP is coupled to NADH oxidation via pyruvate kinase (PK) and lactate dehydrogenase (LDH). With UDPGlc as substrate, UDP was converted to ADP via adenylate kinase and subsequent coupling to PK and LDH reactions. Using this assay, we determined the kinetic parameters of GS and compared them with those obtained with the classical radiochemical method. For this purpose, we improved the expression procedure of A. tumefaciens GS using Escherichia coli BL21(DE3)-RIL cells. This assay allows the continuous monitoring of glycosyltransferase activity using ADPGlc or UDPGlc as sugar-nucleotide donors.     

An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases

Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.     

A unique activity assay for carboxypeptidase A in human serum

Measurement of carboxypeptidase A, one of the pancreatic proteolytic enzymes, in human serum is made possible by a combination of affinity chromatography to isolate and concentrate the enzyme followed by monitoring activity spectrophotometrically with a high-turnover peptide substrate. Concentrations of enzyme in the nanogram-per-milliliter range can be determined with high precision and reliability. Initial clinical application of this method demonstrates no detectable activity in serum from normal individuals, but the enzyme is present in the sera of individuals with pancreatitis.     

Human plasma carboxypeptidase N. Isolation and characterization.

Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield. Isolation was accomplished by chromatography on DEAE-cellulose and on a p-aminobenzoyl-L-arginine-Sepharose 6B affinity column. Carbohydrate accounts for 17% of the weight calculated from its amino acid and carbohydrate composition. The enzyme appears to consist of three subunits of Mr = 83,000, 55,000, and 49,000 and contains a significant amount of bound zinc. Purified enzyme preparations are very sensitive to proteolytic degradation but are stable for at least 3 months at 4 degrees.     

An improved fluorometric assay for DNA

A modification of the fluorometric assay of Kissane and Robins is described. The modified procedure is very accurate, widely applicable, and reasonably easy to use. A standard cell type instead of a standard DNA solution makes the assay universally reproducible. DNA content is calculated by a simple method from the slope of a DNA concentration series. This can be used to detect technique erros.     

Dipeptidyl carboxypeptidase from human seminal plasma.

Dipeptidyl carboxypeptidase (angiotensin I converting enzyme) was purified from human seminal plasma. The apparent relative molecular mass determined by gel filtration on Sephadex G-200 was 330 000. The pI in isoelectric focusing was 4.6--5.0 and the optimum pH 7.7--8.0. The enzyme is activated by chloride. These properties are similar to those reported for the lung enzyme. The specificity is that of a carboxypeptidase releasing dipeptides. A study of different substrates showed the activity to be highest with Z-Leu-Gly-Gly, followed by Z-Phe-His-Leu greater than bradykinin greater than Bz-Gly-Gly-Gly greater than Boc-Phe-Ala-Pro greater than Bz-Gly-His-Leu greater than angiotensin I.     

A potent mercapto bi-product analogue inhibitor for human carboxypeptidase N

The bi-product analogue inhibitor, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, has been synthesized in high yield and exhibits a K_i of 2.0 nM with human plasma carboxypeptidase N. The ease of synthesis and subsequent availability make it an ideal compound to study potentiation of bradykinin and other vasoactive peptides.     

An improved HPLC assay for phosphatidylcholine hydroperoxides (PCOOH) in human plasma with synthetic PCOOH as internal standard

Quantitative and qualitative analyses of 1-palmitoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 16:0/18:2-OOH] and 1-stearoyl-2-linoleoyl-phosphatidylcholine monohydroperoxide [PC 18:0/18:2-OOH] in human plasma were improved by chemiluminescence HPLC using synthetic 1-stearoyl-2-erucoyl-phosphatidylcholine monohydroperoxide (PC 18:0/22:1-OOH) as internal standard. The calibration curves of synthetic PC 16:0/18:2-OOH and PC 18:0/18:2-OOH, obtained by their direct injections with the IS into the HPLC system, were linear throughout the calibration range (10-1000 pmol). Within-day and between-day coefficients of variation were below 8%, and the recoveries were between 84% and 101%. Plasma concentrations of PC 16:0/18:2-OOH and PC 18:0/18:2-OOH were 102+/-59 nM (mean+/-SD) and 36+/-20 nM, respectively, in the 33 healthy volunteers. The present method might help understanding incompletely understood pathway of plasma phosphatidylcholine hydroperoxides.     

An improved glucoseoxidase--peroxidase-coupled assay for -fructofuranosidase activity

An improved glucoseoxidase-peroxidase-coupled assay for the determination of β-fructofuranosidase activity is described. The method makes use of the double effect of Tris (2-amino-2-hydroxymethylpropane-1,3-diol) as an inhibitor of both invertase and contaminating glucosidases. The method is very sensitive and is suitable for routine determinations. The total time needed for a single analysis is less than half an hour.