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At least 343 records · Page 19Linked to original sources
PubMed1988

Fast functional assay of protein C in whole plasma using a snake venom activator: evaluation in patients with congenital and acquired protein C deficiencies.

Both anticoagulant and amidolytic activities of protein C (PC) were measured using a snake venom activator in 4 patients with hereditary PC deficiency, 37 with disseminated intravascular coagulation (DIC), and 30 under stabilized warfarin therapy. The results were compared with those obtained by an immunological assay (ELISA). PC levels measured by different functional and immunological assays were very close in patients with hereditary PC deficiency and DIC. In patients under stable oral anticoagulant therapy, there was no detectable difference between amidolytic activity and antigen levels of PC in each patient plasma, whereas a decrease in anticoagulant activity was much more pronounced. These results indicate that the present activity assays measure PC specifically, and that the snake venom activator is capable of activating both carboxylated and hypocarboxylated forms of PC, but only anticoagulant assay can evaluate the physiological PC function in vitamin K-deficient states.

Biological Assay↗
PubMed1988

A sensitive kinetic assay of serum albumin based on its enzyme-like hydrolytic activity, using a new chromogenic and fluorogenic substrate.

A new albumin assay, based on the unusual enzyme-like activity of the protein that promotes hydrolysis of ester bonds in fatty acid arylesters was designed for clinical routine use. The substrate introduced shows improved analytical wavelengths and is suitable for both photometric and fluorimetric assay. Experiments have been performed with a conventional photometer, a fluorimeter and a Cobas Fara autoanalyzer. Detection limits are as low as 10 micrograms/ml photometrically and 20 ng/ml fluorimetrically. The method provides a sensitive quantitative determination of even minute amounts of albumin in liquid solution, and a simple semiquantitative test may be performed by fixing the dye on a test strip which then is immersed into a sample solution and observing the development of yellow color intensity.

Autoanalysis↗
PubMed1990

A sensitive and specific assay for granulocyte elastase in inflammatory tissue fluid using L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide.

Granulocyte elastase (GE, EC 3.4.21.37) is a key enzyme in tissue injury. To elucidate the role of GE in tissue injury, a new method of measuring GE activity in various inflammatory tissue fluids was developed using diazotization and the chromogenic synthetic substrate, L-pyroglutamyl-L-prolyl-L-valine-p-nitroanilide (S-2482). GE activity demonstrated first order kinetics in the range from 1.9 to 30 U/l. Other proteases, such as pancreatic elastase, trypsin, and chymotrypsin did not hydrolyze S-2484. This assay permits the determination of GE activity with a coefficient of variance less than 7.8% and 95.6 to 105.4% recovery. With this method, hydrolytic GE activity was found to be increased in bronchoalveolar lavage fluid from patients with ARDS or pneumonia, synovial fluid from patients with rheumatoid arthritis, and blister fluid from burn patients.

Blister↗
PubMed1991

Mechanized assay of plasma prekallikrein by activation with Pseudomonas aeruginosa elastase and amidolysis of chromogenic substrate.

An automated assay of plasma prekallikrein is described. Prekallikrein was converted to kallikrein with Pseudomonas aeruginosa elastase, and the hydrolytic activity of kallikrein to H-D-Pro-Phe-Arg-paranitroanilide subsequently measured. The conversion was complete within 8 minutes and the amidolytic activity remained stable at least another 10 min at 37 degrees C. This method worked in plasma deficient in Hageman factor (blood coagulation factor XII). Using anti-prekallikrein antibody and plasma deficient in prekallikrein, the amidolytic activity generated in normal plasma was identified as due to kallikrein. With plasma samples, the coefficients of variation (CV) for multiple measurements within run (n = 10) and between run (n = 10) were as low as 5.0% and 6.6%, respectively, and the minimum measurable concentration of prekallikrein in plasma was 10% of the normal level.

Adult↗
PubMed1994

Glomerular filtration rate and creatinine production in adult icteric patients.

Various methods for serum creatinine determination were compared and validity of the Cockroft-Gault algorithm for calculating creatinine clearance was tested in adult icteric patients. Using conventional Jaffé assays, negative interference is proportional to the serum bilirubin content. Pretreatment of the serum with bilirubin oxidase was more efficient in eliminating bilirubin than pretreatment with potassium ferricyanide. Due to a continued creatine-poor diet and liver dysfunction, erythrocyte creatine levels and creatinine output rate were decreased. Median effect (creatinine equivalent) of non-specific chromogens in the unmodified Jaffé assay was 21 mumol/l (range: 1-108 mumol/l), vs. 19 mumol/l (range: 16-26 mumol/l) for the reference population. In the absence of multi-organ failure, the Cockroft-Gault algorithm could be used for estimating glomerular filtration rate. In patients with multiple organ failure however, we recommend correction for both bilirubin and non-specific chromogens for measuring the serum creatinine concentration.

Adult↗
PubMed1994

Optimized determination of angiotensin I-converting enzyme activity with hippuryl-L-histidyl-L-leucine as substrate.

The determination of the angiotensin I-converting enzyme activity (ACE, kininase II, peptidyldipeptide hydrolase, EC 3.4.15.1) is necessary to control the course and the treatment of sarcoidosis, as well as to monitor the therapeutic use of enzyme inhibitors such as captopril in hypertension or congestive heart failure. Numerous synthetic substrates are known with which to measure the enzyme activity. A discontinuous method using hippuryl-L-histidyl-L-leucine was tested and improved. The cleavage product, hippurate, reacts with cyanuric chloride to give a yellow complex which can be measured at 405 nm using a spectral line photometer. Enzyme activity, kinetic constants and activation energy are dependent on the chloride ion concentration. Optimal test concentrations are 1.1 mol/l potassium chloride and 3.0 mmol/l hippuryl-L-histidyl-L-leucine at pH 8.3. Higher substrate concentrations effect an inhibition of the enzyme reaction. A Michaelis constant of 0.9 mmol/l was found with serum as enzyme source. An activation energy of 57 kJ/mol was obtained from the relation between the logarithm of velocity of enzyme reaction and reciprocal value of absolute temperature. Furthermore, a linear dependence on chloride ion concentration was observed. The histogram of the enzyme activities in sera from 146 healthy volunteers shows a non-gaussian distribution. The reference interval at 25 degrees C is characterized by a median of 24 units/l with the 2.5th and the 97.5th percentiles at 13 units/l and 42 units/l, respectively. The corresponding values at 37 degrees C are 27 units/l and 86 units/l with a median of 48 units/l. No significant sex and age dependence could be found. A potent ACE inhibitor such as captopril leads to a rapid decrease of the enzyme activity within 60 min after oral administration. In the following hours, the enzyme activity slowly increases.

Chromogenic Compounds↗
PubMed1996

Evaluation of endotoxiuria for diagnosis of urinary tract infection after major surgical procedures.

In 148 patients after major surgical procedures urinary endotoxin levels were determined and compared with bacteriological results. The study was designed as a screening study. Urine samples were collected once by suprapubic or transurethral catheters. In a first series of 49 patients urine bacteriology was positive (mainly, Gram-negative rods were found) in 3 cases. However, endotoxin determination was positive in these 3 patients and in a further 10 patients receiving antibiotic therapy for other reasons. Therefore, the following 99 patients were studied also by urinalysis by reagent strips for leukocytes and nitrite. In the second series, 12 urine cultures positive for bacteria were observed. Eleven samples were also endotoxin positive. Five more patients were endotoxin positive and had pathological but unspecific reagent strip results. These patients were treated with antibiotics for other reasons. Patients with candida found in the urine culture (n = 5) were endotoxin negative. Thus, endotoxin determination in urine obtained by suprapubic or transurethral catheters proved to be a very sensitive method for diagnosis of bacterial contamination, even during antibiotic treatment.

Adult↗
PubMed1985

Computerized analysis of enzyme cascade reactions using continuous rate data obtained with an ELISA reader.

Two programs have been written which permit analysis of multiple continuous-rate enzyme-cascade assays conducted with the use of an ELISA spectrophotometer and a synthetic chromogenic substrate. Because the product of the first reaction functions as the enzyme in the second reaction, production of chromophore continuously accelerates and it is the rate of acceleration which serves to measure the rate of the initial reaction in the system. The first program determines the rate of acceleration using linear regression to analyze the reaction curves as a function of the square of time. The second program, using a Simplex algorithm, determines the parameters which establish the assay standard curve by fitting the rate data to the Hill equation. Used together, these programs facilitate the analysis of many kinetic experiments conducted simultaneously.

Chromogenic Compounds↗
PubMed1994

Computer acquisition, analysis and presentation of results from a Cobas Bio analyser.

Two computer programs for Macintosh computers have been developed in the LabVIEW programming language to perform data acquisition, analyses and presentation of results originating from a Cobas Bio analysing machine. One program is very flexible in its human interface, and is intended for research use. The program allows the results to be combined in a variety of ways, and allows the user to generate graphs to highlight the results. The other program is intended for routinely follow-up of analyses of proteases. It only allows the user to follow strict instructions, and only limited flexibility is built-in.

Autoanalysis↗
PubMed1984

Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum: comparative proteolytic activity.

At least four proteolytic activities were detected in the lysates of each of Trichomonas vaginalis, Tritrichomonas foetus, and Trichomitus batrachorum. These were HPAase, a dithiothreitol-dependent activity on hide powder azure; AZCase, a dithiothreitol-dependent activity on azocasein; and two distinct activities towards peptide nitroanilide derivatives--one was optimally active at pH 7 and stimulated by dithiothreitol; the other had no dithiothreitol requirement and was highly active at pH 5. HPAase and AZCase were active over a broad pH range. Overall, with respect to these four activities, T. batrachorum and T. vaginalis were quite similar. In contrast, T. vaginalis and T. foetus differed from one another in several respects, notably the level of HPAase activity and the properties of the dithiothreitol-independent activity. Multiple bands of proteinase activity were demonstrated with each species after electrophoresis of parasite extracts on polyacrylamide gels containing denatured haemoglobin. They appeared optimally at acid pH and in the presence of dithiothreitol. The proteinase band patterns of T. foetus were similarly complex (at least six bands), whereas T. batrachorum gave a much simpler pattern (three bands). The sensitivities to proteinase inhibitors suggested that all the activities were due to cysteine proteinases. The results show that there are some similarities in the proteolytic activities of all three trichomonad species, and that the two parasites of the urinogenital tracts of mammals possess additional features in common.

Animals↗
PubMed1986

Evolutionary conservation of the substrate-binding cleft of phosphoglycerate kinases.

The primary structures of six phosphoglycerate kinases (PGKs) are known: three from mammals, one from yeast, and two from trypanosomes. Comparison of the amino acid sequence of these enzymes reveals 154 invariant positions out of 392 positions in the aligned sequences. Most of the conserved positions fall into the twelve beta-sheets and adjacent peptide regions that form the inner loops surrounding the ATP and 3-phosphoglycerate-binding cleft. The homology between mammalian and yeast PGKs is greater than 94% for the inner-loop region, even though the overall homology is less than 65%. Trypanosome PGK has only 44% overall homology with the mammalian enzyme, but shows 74% homology in the inner-loop region. Trypanosome PGK contains a polypeptide segment in its N-terminal domain that is transposed in comparison with the other species.

Amino Acid Sequence↗
PubMed1995

Monomeric human cathepsin E.

Cathepsin E is a homodimer, consisting of two monomers linked by an inter-molecular disulphide bond. The cysteine residue involved is located near to the N-terminus of the mature proteinase. By mutating this residue to alanine, a monomeric form of human cathepsin E was engineered and purified. The activity of the resultant enzyme was not altered significantly (in terms of its ability to hydrolyse two chromogenic peptide substrates; and its susceptibility to inhibition by pepstatin). However, the stability of the mutant enzyme to alkaline pH and to temperature was markedly reduced.

Amino Acid Sequence↗
PubMed1990

Isolation of two hemorrhagic toxins from Crotalus basiliscus basiliscus (Mexican west coast rattlesnake) venom and their effect on blood clotting and complement.

1. Two hemorrhagic toxins of mol. wt 27,000 (B1) and 27,500 (B2) and pI 9.8 and 5.2 respectively were isolated from Crotalus basiliscus venom. 2. The two proteinases did not cross-react antigenically. 3. Both toxins caused hemorrhage in mice and each was capable of hydrolyzing hide power azure, casein, collagen and fibrin. 4. B1 hydrolyzed the A alpha, B beta and gamma chains of fibrinogen. B2 hydrolyzed the A alpha and B beta chains of fibrinogen, but not the gamma chain. 5. Both proteinases inactivated guinea pig complement.

Animals↗
PubMed1987

The chromogenicity and quantitation of apoB-100 and apoB-48 of human plasma lipoproteins on analytical SDS gel electrophoresis.

ApoB-100 and apoB-48 may be readily resolved in 3.3% sodium dodecyl sulphate-polyacrylamide gels. This study has characterized the relative chromogenicities (staining intensity/micrograms protein) of human apoB-100 and apoB-48 in various lipoprotein classes with Coomassie Brilliant Blue (R250) upon SDS-PAGE. The relation between dye uptake and the mass of each apoB species in any lipoprotein preparation, was linear at least within the concentration range of total apoprotein B which is optimally resolved in these gels (20-50 micrograms total apoprotein B), and was a function of the density of the particular lipoprotein fraction under investigation. There was a constant and characteristic difference between the chromogenicity for apoB-100 and that for apoB-48 as determined from the slopes of their respective chromogenicity curves. The slope of the lines describing staining intensity vs. protein mass for both apoB-100 and apoB-48 decreased as the density of the lipoprotein fraction increased. The slope of the line for apoB-100 was steeper than that for apoB-48 (i.e. chromogenicity apoB-100 greater than apoB-48) in all lipoprotein fractions where both were present. The relationship between the slopes of the lines for apoB-100 and apoB-48 was constant regardless of the density of the lipoprotein fraction. The chromogenicity curves for apoB-100 and for apoB-48 obtained when lipoprotein samples were applied to gels in concentrations conventionally used for this technique (i.e. 20-100 micrograms total apoB/gel) did not extrapolate to the same point on the ordinate, which precludes the use of a simple ratio or "chromogenicity factor" to describe their relative chromogenicities over this concentration range, Hence, a novel approach was developed to determine the relative mass of apoB-100/apoB-48 in lipoprotein samples, based on their staining characteristics in SDS-PAGE.

Apolipoprotein B-100↗
PubMed1982

Miniaturization of beta-galactosidase immunoassays using chromogenic and fluorogenic substrates.

Enzyme immunoassay techniques are widely use to quantify various antigens and antibodies. The final step of these techniques (i.e. enzyme reaction) may be carried out in several ways (e.g. chromogenic, fluorogenic, or radioactive substrate and thermometric measurement). This paper compares the effectiveness of the chromogenic and the fluorogenic substrates in the beta-galactosidase immunoassay. Using microtitration plates (150 microliter samples) coated with anti-human IgE, and anti-human IgE labeled with E. coli beta-galactosidase, the lowest concentrations of IgE that one could detect employing either the chromogenic (o-nitrophenyl-beta-D-galactopyranoside) or the fluorogenic (4-methyl-umbelliferyl-beta-D-galactopyranoside) substrate were determined. It was found that both substrates were almost equally effective in measuring the lowest concentration of IgE (0.075-0.13 IU/ml) under the optimal conditions. But, using fluorogenic substrate and suitable apparatuses the enzyme immunoassay can be miniaturized. Thus by using decreasing volumes of reagents, progressively smaller amounts of antigen were quantified: as the sample volumes were reduced from 150 to 10 microliter and finally to 0.3 microliter a progressive decrease from 7 x 107 molecules of IgE to 2.9 x 107 molecules and to 1.5 x 106 molecules was observed. The corresponding lowest detection limits were 0.075 IU/ml, 0.46 IU/ml and 0.8 IU/ml.

Animals↗
PubMed1984

Microassay for the photometric quantitation of cell-associated plasminogen activator using a chromogenic tripeptide substrate.

Plasminogen activator (PA), a neutral protease whose primary function is to convert plasminogen to plasmin, is produced by various cells including macrophages, monocytes, endothelial cells, and tumor cells. This study reports the use of the chromogenic tripeptide substrate D-Val-Leu-Lys-p-nitroanilide (S-2251) and an automated microtiter plate reader spectrophotometer for the determination of PA activity in cells and fluids. There was a linear relationship between the time of incubation at 37 degrees C and the square root of the absorbance measured at 405 nm when urokinase was incubated with the substrate in the presence of plasminogen. There was no activity in the absence of plasminogen. The slopes of the lines (square root A 405/time) were directly related to the concentrations of urokinase, up through 0.05 CTA units. Using this assay, we determined the cellular activity of PA in human promyelocytic cells HL-60 (1.33 +/- 0.12 CTA units/mg), human monocytoid cells U937 (1.27 +/- 0.12 CTA units/mg), mouse myeloid leukemia cells RFM/UN (0.70 +/- 0.07 CTA units/mg), freshly isolated normal human monocytes (0.00 +/- 0.00 CTA units/mg), and human monocytes after 7 days in culture (5.66 +/- 0.38 CTA units/mg). There was a variable amount of activity expressed in freshly isolated cells or cell lysates of peritoneal macrophages from normal mice, or mice that had gotten intraperitoneal injections of peptone, thioglycollate, or NaIO4, but after 24 or 48 h of culture, these activities, in general, increased. Using this assay, PA levels in the euglobulin precipitates from human plasma prepared without venous occlusion (0.03 +/- 0.02 CTA units/mg protein) or after 5 min of venous occlusion of the arm (0.18 +/- 0.01 CTA units/mg) were comparable to those reported by others using different assays. Thus, this represents a simple, rapid, accurate assay of PA that should be useful to those in immunology, cell biology, and clinical medicine.

Animals↗
PubMed1986

Luminescent visualization of antigens on blots.

Proteins separated by SDS gel electrophoresis and transferred to a nitrocellulose sheet can be visualised by 'probing' with peroxidase-linked reagents which are detected by luminescence. A modified luminescent substrate is described containing 4-methylumbelliferone which enhances light emission four-fold. Using the modified substrate, luminescent detection was found to be more sensitive than chromogenic detection of peroxidase using 4-chloro-1-napthol. The new technique was used in conjunction with the immunoblot method to demonstrate antigenic differences between rat and mouse erythrocytes.

Animals↗