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Title: Detection of an azido-(/sup 14/C)-atrazine labeled protein transferred to nitrocellulose paper

Abstract

An electrophoretically similar protein in spinach and maize can be covalently labeled with azido-(/sup 14/C)-atrazine and separated by 10-18% gradient LDS-PAGE. The protein profile can be transferred to nitrocellulose paper (ncp) by western blotting. The ncp containing the protein profile is sliced into 2 mm slices and counted with liquid scintillation. The labeled protein migrates as a diffuse band with a Mr of 34 kD. This band migrates at a higher Mr (40 kD) under different gel conditions. The ncp dissolves in the organic scintillation cocktail thus providing a more sensitive and quantitative detection of the /sup 14/C. This technique allows the simultaneous immunological and radiochemical identification of many electrophoretically separable proteins.

Authors:
; ;
Publication Date:
Research Org.:
Dept. of Biological Sciences, Denver, CO
OSTI Identifier:
5098188
Alternate Identifier(s):
OSTI ID: 5098188
Resource Type:
Conference
Resource Relation:
Journal Name: Plant Physiol., Suppl.; (United States); Journal Volume: 80:4; Conference: Annual meeting of the American Society of Plant Physiologists, Baton Rouge, LA, USA, 8-12 Jun 1986; Other Information: C45.00*
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; MAIZE; PROTEINS; LABELLING; SPINACH; CARBON 14 COMPOUNDS; ELECTROPHORESIS; NITROCELLULOSE; SEPARATION PROCESSES; CARBOHYDRATES; CELLULOSE ESTERS; CEREALS; CHEMICAL EXPLOSIVES; ESTERS; EXPLOSIVES; FOOD; GRASS; LABELLED COMPOUNDS; NITRIC ACID ESTERS; ORGANIC COMPOUNDS; PLANTS; POLYSACCHARIDES; SACCHARIDES; VEGETABLES 550200* -- Biochemistry

Citation Formats

Ivey, S., Metz, J.G., and Berg, S.P. Detection of an azido-(/sup 14/C)-atrazine labeled protein transferred to nitrocellulose paper. United States: N. p., 1986. Web.
Ivey, S., Metz, J.G., & Berg, S.P. Detection of an azido-(/sup 14/C)-atrazine labeled protein transferred to nitrocellulose paper. United States.
Ivey, S., Metz, J.G., and Berg, S.P. Tue . "Detection of an azido-(/sup 14/C)-atrazine labeled protein transferred to nitrocellulose paper". United States. doi:.
@article{osti_5098188,
title = {Detection of an azido-(/sup 14/C)-atrazine labeled protein transferred to nitrocellulose paper},
author = {Ivey, S. and Metz, J.G. and Berg, S.P.},
abstractNote = {An electrophoretically similar protein in spinach and maize can be covalently labeled with azido-(/sup 14/C)-atrazine and separated by 10-18% gradient LDS-PAGE. The protein profile can be transferred to nitrocellulose paper (ncp) by western blotting. The ncp containing the protein profile is sliced into 2 mm slices and counted with liquid scintillation. The labeled protein migrates as a diffuse band with a Mr of 34 kD. This band migrates at a higher Mr (40 kD) under different gel conditions. The ncp dissolves in the organic scintillation cocktail thus providing a more sensitive and quantitative detection of the /sup 14/C. This technique allows the simultaneous immunological and radiochemical identification of many electrophoretically separable proteins.},
doi = {},
journal = {Plant Physiol., Suppl.; (United States)},
number = ,
volume = 80:4,
place = {United States},
year = {Tue Apr 01 00:00:00 EST 1986},
month = {Tue Apr 01 00:00:00 EST 1986}
}

Conference:
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  • The metabolism of [2,4,6-{sup 14}C]-atrazine in field-grown sugarcane and sorghum produced several dechlorinated, dealkylated, and/or glutathione metabolites. These metabolites were identified by gas chromatography-mass spectrometry and electrospray-tandem mass spectrometry. The subsequent metabolism of glutathione adducts of atrazine and N-dealkylated atrazine to novel metabolites were studied. Lanthionine, lanthionine sulfoxide, glucose-thiolactate and glutamine adducts of atrazine are examples of metabolites that we postulate arise from a common intermediate metabolite of atrazine, i.e., atrazine-glutathione adduct at position 2. The mass spectra as well as the postulated metabolic pathways will be discussed. The identification of such metabolites at trace levels in plant tissues weremore » made possible by the coupling of the radioactive detector and electrospray interface on-line to the tandem mass spectrometer.« less
  • Using unperturbed-structure soil columns, placed under field conditions for a 1-year period, we have evaluated the possibilities of penetration into the soil of various /sup 14/C ring-labeled s-triazinic compounds: atrazine, chlorinated derivatives, and hydroxyatrazine. We have simultaneously examined the possibilities of formation of bound residues for each of the compounds. 2-Cl-4,6-Diaminoatrazine appears to be the product most apt to form bound residues but this reaction is paralleled by a rapid loss of total residues. On the other hand, hydroxyatrazine is a strongly adsorbed product which is lost from the soil into the atmosphere only slowly and forms practically no boundmore » residues.« less
  • By the use of unperturbed-structure soil columns placed under field conditions, we have evaluated the migration of various /sup 14/C ring-labeled s-triazinic compounds (atrazine, its chlorinated derivatives, hydroxyatrazine) into leached water over a 1-year period. The results obtained by determination of the leached radioactivity allow us to establish a classification of the mobility for each product and its possibility of contaminating infiltration water in the soil. Qualitative analysis of the leached residues confirms the predominant presence of deethylated atrazine.
  • Sugarcane and sorghum plants were grown in separate field plots and treated with [2,4,6-{sup 14}C]-Atrazine (according to standard agricultural practices and at levels approximating the maximum usage rate) in partial fulfillment of EPA registration requirements. Sugarcane leaves were collected just before the final (fourth) test material application and at final harvest; canes were collected only at final harvest. Atrazine and a total of 20 metabolites of atrazine, accounting for 45.1% of the total radioactive residues, were isolated and characterized from prefourth application sugarcane leaves. Sorghum forage samples were collected 30 days after treatment (30 DAT), and at silage stage; maturemore » fodder and grain were collected at final harvest. Two additional metabolites of atrazine were isolated and characterized from 30 DAT sorghum. Flowcharts describing the extraction and fractionation procedures used for isolation and purification of selected metabolites will be presented. The mass spectra as well as proposed metabolic pathways for these metabolites will be presented in an accompanying abstract.« less
  • In order to determine the rate of transfer and the nature of the atrazine residues present in milk, lactating dairy cattle were treated with atrazine at three concentrations, 0.764 ppm, 0.0747 ppm and 0.0085 ppm (dry weight of food consumed). The concentrations were selected to bridge the gap between the concentration used for EPA metabolism studies (10 ppm) and the potential exposure level of dairy cattle to atrazine and its chlorotriazine metabolites through feed. The cattle were dosed following the morning milking for nine consecutive days with a single capsule bolus of {sup 14}C-atrazine. Milk was collected twice daily andmore » aliquots of each milking and the individual cow`s daily pool of milk were analyzed by liquid scinitllation counting (LSC). The concentrations of {sup 14}C-residues in the milk plateaued on approximately day 3 and the mean {sup 14}C-atrazine levels in milk were 11.2 ppb, 1.13 ppb and 0.152 ppb for the high, middle and low dosed animals, respectively. The transfer of radioactive level of exposure to {sup 14}C-atrazine. The nature of the residues in milk were determined by extracting milk samples and analysis by HPLC, TLC or Aminex chromatography. Diaminchlorotriazine was the only chlorinated metabolite in the milk, constituting approximately 65% to 75% of the total radioactive residues (TRR).« less