Urine contains an inhibitor for turbidimetric determinations of protein.

Abstract A greatly simplified radioimmunoassay for bradykinin in human plasma is described. Current techniques require multiple chromatographic steps or extraction procedures with analytical recoveries of bradykinin of often less than 60%. We present a method in which bradykinin is separated from components of higher relative molecular mass (including kininogens) in a single step, by use of a column of Sephadex G-25 medium (PD-10). The mean analytical recovery of tritiated bradykinin added to plasma is 85.5% (SD, 3.5%). The sensitivity of this radioimmunoassay is 25 pg per assay tube, equivalent to 125 ng per liter of plasma. Twenty to 30 blood samples may be completely processed and assayed within 6 h. As determined with this technique, concentrations of bradykinin in plasma from apparently normal subjects ranged from 2.5 to 5.2 microgram/L (mean 4.2, SD 1.1 microgram/L); these values are consistent with previously reported normal values.

Download Full-text

New micro-turbidimetric method for determination of protein in cerebrospinal fluid and urine.

Clinical Chemistry ◽

10.1093/clinchem/25.7.1317 ◽

1979 ◽

Vol 25 (7) ◽

pp. 1317-1319 ◽

Cited By ~ 81

Author(s):

J Iwata ◽

O Nishikaze

Keyword(s):

Cerebrospinal Fluid ◽

Gamma Globulin ◽

Sulfosalicylic Acid ◽

Trichloracetic Acid ◽

Micro Scale ◽

Turbidimetric Method ◽

Classic Method ◽

Benzethonium Chloride ◽

Higher Sensitivity

Abstract We report a new micro-scale (0.1-mL sample) turbidimetric method for determination of protein by use of benzethonium chloride in alkali. The method is highly specific for protein, has a higher sensitivity than the classic method of Lowry et al., and shows satisfactory reproducibility and recovery. The turbidity produced in our method is the same for albumin and gamma-globulin and is more stable than in Meulemans' method (in which sulfosalicylic acid is used) or in the method of Bossak et al. (in which trichloracetic acid is used). In contrast to Pesce and Strande's method, there is no manipulative loss of protein.

Download Full-text

Simplified radioimmunoassay of bradykinin in human plasma.

Clinical Chemistry ◽

10.1093/clinchem/26.3.429 ◽

1980 ◽

Vol 26 (3) ◽

pp. 429-432 ◽

Cited By ~ 3

Author(s):

P S Verma ◽

P E Lorenz ◽

G E Sander

Keyword(s):

Molecular Mass ◽

Human Plasma ◽

Normal Values ◽

Normal Subjects ◽

Single Step ◽

Relative Molecular Mass ◽

Blood Samples ◽

Analytical Recovery ◽

Assay Tube ◽

The Mean

Abstract A greatly simplified radioimmunoassay for bradykinin in human plasma is described. Current techniques require multiple chromatographic steps or extraction procedures with analytical recoveries of bradykinin of often less than 60%. We present a method in which bradykinin is separated from components of higher relative molecular mass (including kininogens) in a single step, by use of a column of Sephadex G-25 medium (PD-10). The mean analytical recovery of tritiated bradykinin added to plasma is 85.5% (SD, 3.5%). The sensitivity of this radioimmunoassay is 25 pg per assay tube, equivalent to 125 ng per liter of plasma. Twenty to 30 blood samples may be completely processed and assayed within 6 h. As determined with this technique, concentrations of bradykinin in plasma from apparently normal subjects ranged from 2.5 to 5.2 microgram/L (mean 4.2, SD 1.1 microgram/L); these values are consistent with previously reported normal values.

Download Full-text

Purification of an alkaline phosphatase-lipoprotein-X complex.

Clinical Chemistry ◽

10.1093/clinchem/26.5.604 ◽

1980 ◽

Vol 26 (5) ◽

pp. 604-608 ◽

Cited By ~ 9

Author(s):

R N Weijers ◽

H Kruijswijk ◽

J M Baak

Keyword(s):

Alkaline Phosphatase ◽

Affinity Chromatography ◽

Molecular Mass ◽

Gel Filtration ◽

Relative Molecular Mass ◽

Agarose Gel ◽

Extrahepatic Cholestasis ◽

Abnormal Position ◽

Alkaline Phosphatase Isoenzyme ◽

Isoenzyme Patterns

Abstract An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

Download Full-text

Cardiac Troponin T Circulates in the Free, Intact Form in Patients with Kidney Failure

Clinical Chemistry ◽

10.1373/clinchem.2005.062307 ◽

2006 ◽

Vol 52 (3) ◽

pp. 414-420 ◽

Cited By ~ 47

Author(s):

Michael N Fahie-Wilson ◽

David J Carmichael ◽

Michael P Delaney ◽

Paul E Stevens ◽

Elizabeth M Hall ◽

...

Keyword(s):

Molecular Mass ◽

Kidney Failure ◽

Cardiac Troponin ◽

Troponin T ◽

Gel Filtration ◽

Serum Prolactin ◽

Relative Molecular Mass ◽

Cardiac Troponin T ◽

Coronary Syndrome ◽

Esrd Patients

Abstract Background: The clinical significance of the increased concentrations of cardiac troponins observed in patients with end stage renal disease (ESRD) in the absence of an acute coronary syndrome (ACS) is controversial. One proposed explanation is that immunoreactive fragments of cardiac troponin T (cTnT) accumulate in ESRD. We used gel-filtration chromatography (GFC) to ascertain whether fragments of cTnT, which could cross-react in the commercial diagnostic immunoassay (Roche Diagnostics), were the cause of the increased cTnT in the serum of patients with ESRD. Methods: We subjected sera from ESRD patients (n = 21) receiving dialysis and having increased cTnT concentrations to size-separation GFC. We detected cTnT in the chromatography fractions by use of the same antibodies used in the commercial assay for serum cTnT. Results: In all patients, cTnT immunoreactivity eluted as a major, homogeneous peak in an identical position between the peaks of serum prolactin [relative molecular mass (Mr) 23 000] and albumin (Mr 67 000): the elution pattern of cTnT in samples obtained from ACS patients was identical to that of the ESRD patients. There was no evidence that low–molecular-mass (Mr <23 000) cTnT fragments were the cause of the increased cTnT in the patients studied. Conclusions: The form of cTnT observed in the serum of patients with kidney failure and immunoreactive in the diagnostic assay is predominantly the free intact form, as in patients with ACS. Our data are consistent with the view that circulating cTnT in renal failure reflects cardiac pathology.

Download Full-text

A squid dynein isoform promotes axoplasmic vesicle translocation.

The Journal of Cell Biology ◽

10.1083/jcb.109.5.2379 ◽

1989 ◽

Vol 109 (5) ◽

pp. 2379-2394 ◽

Cited By ~ 16

Author(s):

S P Gilbert ◽

R D Sloboda

Keyword(s):

Molecular Mass ◽

Gel Filtration ◽

Relative Molecular Mass ◽

Dependent Manner ◽

Latex Beads ◽

Organelle Motility ◽

Punctate Pattern ◽

Bidirectional Movement ◽

Mgatpase Activity ◽

Peak Fraction

Axoplasmic vesicles that translocate on isolated microtubules in an ATP-dependent manner have an associated ATP-binding polypeptide with a previously estimated relative molecular mass of 292 kD (Gilbert, S. P., and R. D. Sloboda. 1986. J. Cell Biol. 103:947-956). Here, data are presented showing that this polypeptide (designated H1) and another high molecular mass polypeptide (H2) can be isolated in association with axoplasmic vesicles or optic lobe microtubules. The H1 and H2 polypeptides dissociate from microtubules in the presence of MgATP and can be further purified by gel filtration chromatography. The peak fraction thus obtained demonstrates MgATPase activity and promotes the translocation of salt-extracted vesicles (mean = 0.87 microns/s) and latex beads (mean = 0.92 microns/s) along isolated microtubules. The H1 polypeptide binds [alpha 32P]8-azidoATP and is thermosoluble, but the H2 polypeptide does not share these characteristics. In immunofluorescence experiments with dissociated squid axoplasm, affinity-purified H1 antibodies yield a punctate pattern that corresponds to vesicle-like particles, and these antibodies inhibit the bidirectional movement of axoplasmic vesicles. H2 is cleaved by UV irradiation in the presence of MgATP and vanadate to yield vanadate-induced peptides of 240 and 195 kD, yet H1 does not cleave under identical conditions. These experiments also demonstrate that the actual relative molecular mass of the H1 and H2 polypeptides is approximately 435 kD. On sucrose density gradients, H1 and H2 sediment at 19-20 S, and negatively stained samples reveal particles comprised of two globular heads with stems that contact each other and extend to a common base. The results demonstrate that the complex purified is a vesicle-associated ATPase whose characteristics indicate that it is a squid isoform of dynein. Furthermore, the data suggest that this vesicle-associated dynein promotes membranous organelle motility during fast axoplasmic transport.

Download Full-text

Purification of an alkaline phosphatase-lipoprotein-X complex.

Clinical Chemistry ◽

10.1093/clinchem/26.5.0604 ◽

1980 ◽

Vol 26 (5) ◽

pp. 604-608

Author(s):

R N Weijers ◽

H Kruijswijk ◽

J M Baak

Keyword(s):

Alkaline Phosphatase ◽

Affinity Chromatography ◽

Molecular Mass ◽

Gel Filtration ◽

Relative Molecular Mass ◽

Agarose Gel ◽

Extrahepatic Cholestasis ◽

Abnormal Position ◽

Alkaline Phosphatase Isoenzyme ◽

Isoenzyme Patterns

Abstract An alkaline phosphatase isoenzyme was observed in an abnormal position in the agar-agarose gel pattern of sera from several patients suffering from intrahepatic and extrahepatic cholestasis. We purified this isoenzyme, by gel filtration and affinity chromatography, from the serum of a patient suffering from extrahepatic cholestasis. Analysis demonstrated an alkaline phosphatase-lipoprotein-X complex with a relative molecular mass of at least 669,000. We discuss the interpretation of alkaline phosphatase isoenzyme patterns produced by different techniques.

Download Full-text

Extraction of polyethylene glycols with dicarbolide solutions in nitrobenzene

Collection of Czechoslovak Chemical Communications ◽

10.1135/cccc19901959 ◽

1990 ◽

Vol 55 (8) ◽

pp. 1959-1967 ◽

Cited By ~ 2

Author(s):

Petr Vaňura ◽

Pavel Selucký

Keyword(s):

Polyethylene Glycol ◽

Molecular Mass ◽

Extraction Constant ◽

Metal Extraction ◽

Polyethylene Glycols ◽

Relative Molecular Mass ◽

Published Data ◽

Average Molecular Mass ◽

Peg 400 ◽

Extraction Constants

The extraction of polyethylene glycol of average molecular mass 400 (PEG 400) with dicarbolide solution in nitrobenzene and of longer-chain polyethylene glycol, of average molecular mass 1 500 (PEG 1 500), with chlorinated dicarbolide solution in nitrobenzene was studied. During the extraction of PEG 400, the polyethylene glycol solvates the Horg+ ion in the organic phase giving rise to the HLorg+ species (L is polyethylene glycol). The obtained value of the extraction constant Kex(HLorg+) = 933 is consistent with published data of metal extraction. Extraction of PEG 1 500 was treated applying the simplified assumption that the thermodynamic behaviour of PEG 1 500 is the same as that of n molecules of polyethylene glycol with relative molecular mass 1 500/n, each solvating one cation. For this model, the value of n = 3.2 ± 1.1 and the values of the extraction constants of the HL1/n,org+ and HL2/n,org+ species were obtained by using the adapted program LETAGROP. This value of n is consistent with published extraction data in the presence of polyethylene glycol with a relative molecular mass from 200 to 1 000.

Download Full-text

Modified Assay Medium for the Turbidimetric Assay of Chlortetracycline in Feeds

Journal of AOAC INTERNATIONAL ◽

10.1093/jaoac/59.3.536 ◽

1976 ◽

Vol 59 (3) ◽

pp. 536-539

Author(s):

Hussein S Ragheb

Keyword(s):

Growth Rate ◽

Excellent Agreement ◽

Test Organism ◽

Plate Method ◽

Assay Sensitivity ◽

Assay Medium ◽

Turbidimetric Method ◽

Turbidimetric Assay ◽

Commercial Feeds ◽

Lower Recovery

Abstract In previous experiments, the turbidimetric method for determining chlortetracycline-HCI (CTC-HCl) in feeds showed lower recovery than the AOAC plate method. Although the addition of vitamins to the turbidimetric medium improved results, values by the turbidimetric method remained about 10% lower than by the plate method. A modified (1.7 × the weight recommended by the manufacturer) turbidimetric assay medium decreased assay sensitivity but did not significantly change the slope of S. aureus response to CTC-HCl. There was no evidence that vitamin fortification of the modified medium had any significant effect on the growth rate of test organism. Examination of about 100 samples of commercial feeds containing CTC-HCl showed excellent agreement in results between the turbidimetric and plate methods.

Download Full-text

Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway

Biochemical Journal ◽

10.1042/bj20041053 ◽

2005 ◽

Vol 387 (1) ◽

pp. 271-280 ◽

Cited By ~ 58

Author(s):

Seonghun KIM ◽

Sun Bok LEE

Keyword(s):

Molecular Mass ◽

Gel Filtration ◽

Maximal Activity ◽

Genome Database ◽

Sds Page ◽

Key Enzyme ◽

Bivalent Metal Ions ◽

Ammonium Sulphate Fractionation ◽

Identification And Characterization

The extremely thermoacidophilic archaeon Sulfolobus solfataricus utilizes D-glucose as a sole carbon and energy source through the non-phosphorylated Entner–Doudoroff pathway. It has been suggested that this micro-organism metabolizes D-gluconate, the oxidized form of D-glucose, to pyruvate and D-glyceraldehyde by using two unique enzymes, D-gluconate dehydratase and 2-keto-3-deoxy-D-gluconate aldolase. In the present study, we report the purification and characterization of D-gluconate dehydratase from S. solfataricus, which catalyses the conversion of D-gluconate into 2-keto-3-deoxy-D-gluconate. D-Gluconate dehydratase was purified 400-fold from extracts of S. solfataricus by ammonium sulphate fractionation and chromatography on DEAE-Sepharose, Q-Sepharose, phenyl-Sepharose and Mono Q. The native protein showed a molecular mass of 350 kDa by gel filtration, whereas SDS/PAGE analysis provided a molecular mass of 44 kDa, indicating that D-gluconate dehydratase is an octameric protein. The enzyme showed maximal activity at temperatures between 80 and 90 °C and pH values between 6.5 and 7.5, and a half-life of 40 min at 100 °C. Bivalent metal ions such as Co2+, Mg2+, Mn2+ and Ni2+ activated, whereas EDTA inhibited the enzyme. A metal analysis of the purified protein revealed the presence of one Co2+ ion per enzyme monomer. Of the 22 aldonic acids tested, only D-gluconate served as a substrate, with Km=0.45 mM and Vmax=0.15 unit/mg of enzyme. From N-terminal sequences of the purified enzyme, it was found that the gene product of SSO3198 in the S. solfataricus genome database corresponded to D-gluconate dehydratase (gnaD). We also found that the D-gluconate dehydratase of S. solfataricus is a phosphoprotein and that its catalytic activity is regulated by a phosphorylation–dephosphorylation mechanism. This is the first report on biochemical and genetic characterization of D-gluconate dehydratase involved in the non-phosphorylated Entner–Doudoroff pathway.

Download Full-text