A study of sensitized lanthanide luminescence in an engineered calcium-binding protein.
AuthorsClark ID, MacManus JP, Banville D, Szabo AG
JournalAnal Biochem
PubMed ID8489002
'In this study, the CD loop of the Ca(2+)-binding protein oncomodulin was replaced by a high-affinity, metal-binding sequence that was found to reverse the order of fill of the two sites in the protein. A cysteine was included at position 7 of this sequence, i.e., DKNADGCIEFEE. The cysteine allowed covalent ... More
Self-association of Ca(2+)-binding peptides induced by lanthanide ions: a fluorescence study.
AuthorsClark ID, Brown CM, Sikorska-Walker M, MacManus JP, Szabo AG
JournalAnal Biochem
PubMed ID8238905
'In this study a Ca(2+)-binding 14mer peptide was synthesized with the sequence GDKNADGCIEFEEL, allowing covalent attachment of sulfhydryl-reactive fluorescent molecules at position 7 of the 12-residue, metal-binding loop (underlined). This provided the opportunity to select donor molecules with suitable spectral characteristics for sensitized excitation of chelated terbium (Tb3+) or europium ... More
High-performance liquid chromatography with chemiluminescence detection of serum levels of pre-column derivatized fluoropyrimidine compounds.
AuthorsYoshida S, Urakami K, Kito M, Takeshima S, Hirose S
JournalJ Chromatogr
PubMed ID2148941
'7-(Diethylamino)-3-[4-[iodoacetyl)amino)phenyl]-4-methylcoumarin (DCIA) and 4-(bromomethyl)-7-methoxycoumarin have been evaluated as fluoropyrimidine-derivatizing agents to be detected using peroxyoxalate chemiluminescence with high-performance liquid chromatography. The derivatization procedure required only one step. No chemiluminescence was observed from the bromo derivatives, and the detection limits of fluoropyrimidine compounds derivatized with the iodo compound and detected with ... More
New fluorochromes for thiols: maleimide and iodoacetamide derivatives of a 3-phenylcoumarin fluorophore.
AuthorsSippel TO
JournalJ Histochem Cytochem
PubMed ID7019305
N-(4-(7 - Diethylamino - methylcoumarin - 3 yl)phenyl)maleimide (CPM) and the corresponding iodoacetamide are described. When applied to sections at pH 6 and 9, respectively, the two fluorogens are very similar in their reactivity and selectivity toward thiols with which they form adducts having much the same bright blue fluorescence. ... More
Detection of calcium binding proteins on polyacrylamide gels using time-resolved lanthanide luminescence photography.
AuthorsHill IE, Hogue CW, Clark ID, MacManus JP, Szabo AG
JournalAnal Biochem
PubMed ID7513974
Methods were developed for using the luminescent lanthanides Tb3+ and Eu3+ for the specific staining of calcium-binding proteins, as well as the nonspecific staining of proteins, on polyacrylamide gels. These methods involve equilibration of the gel after electrophoresis in solutions containing the appropriate lanthanide and a weak competitive chelating agent, ... More
Movement of tRNA but not the nascent peptide during peptide bond formation on ribosomes.
AuthorsOdom OW, Picking WD, Hardesty B
JournalBiochemistry
PubMed ID1703007
The results from experiments involving nonradiative energy transfer indicate that a fluorescent probe on the 5'-end of tRNA(Phe) moves more than 20 A towards probes on ribosomal protein L1 as a peptide bond is formed during the peptidyl transferase reaction on Escherichia coli ribosomes. The peptide itself moves no more ... More
Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein.
AuthorsFernandes F, Loura LM, Koehorst R, Spruijt RB, Hemminga MA, Fedorov A, Prieto M,
JournalBiophys J
PubMed ID15240469
Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4'iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the ... More
Fluorescence study of the topology of messenger RNA bound to the 30S ribosomal subunit of Escherichia coli.
AuthorsCzworkowski J, Odom OW, Hardesty B
JournalBiochemistry
PubMed ID2029524
Short RNAs (25-36 nucleotides in length) with sequences of the translational initiation region of bacteriophage R17 protein A mRNA were produced by chemical and in vitro transcription techniques and labeled at their 5' or 3' ends with fluorescent probes. The interaction of these labeled RNAs with the 30S subunit of ... More
Protein-protein and protein-DNA interactions at the bacteriophage T4 DNA replication fork. Characterization of a fluorescently labeled DNA polymerase sliding clamp.
The T4 DNA polymerase holoenzyme is composed of the polymerase enzyme complexed to the sliding clamp (the 45 protein), which is loaded onto DNA by an ATP-dependent clamp loader (the 44/62 complex). This paper describes a new method to directly investigate the mechanism of holoenzyme assembly using a fluorescently labeled ... More
Flow cytometry techniques for studying cellular thiols.
AuthorsDurand RE, Olive PL
JournalRadiat Res
PubMed ID6193555
Cellular thiols, and especially glutathione, act as scavenger nucleophiles and can protect against toxicity, mutagenicity, or transformation by ionizing radiation and many carcinogens. Development of a rapid assay to quantitate the cellular content of thiols could thus be useful in assessing or predicting cellular risk to damage. Several fluorescent thiol-reactive ... More
Novel fluorescent phospholipids for assays of lipid mixing between membranes.
AuthorsSilvius JR, Leventis R, Brown PM, Zuckermann M
JournalBiochemistry
PubMed ID3663589
A series of fluorescent phospholipids has been synthesized, by a general and versatile procedure, with various fluorescent groups attached to the methyl-terminal half of one acyl chain in an otherwise normal phospholipid structure. Phospholipids labeled with (dialkylamino)coumarin moieties, and to a slightly lesser extent those labeled with a bimane group, ... More
Active-site-selective labeling of blood coagulation proteinases with fluorescence probes by the use of thioester peptide chloromethyl ketones. II. Properties of thrombin derivatives as reporters of prothrombin fragment 2 binding and specificity of the labeling approach for other proteinases.
AuthorsBock PE
JournalJ Biol Chem
PubMed ID1634536
The behavior of an array of fluorescent human alpha-thrombin derivatives in reporting binding of the fragment 2 domain of prothrombin was characterized as a representative application of the active-site-selective labeling approach to studies of blood coagulation proteinase regulatory interactions. An array of 16 thrombin derivatives was prepared by affinity labeling ... More
Use of 50 S-binding antibiotics to characterize the ribosomal site to which peptidyl-tRNA is bound.
AuthorsOdom OW, Hardesty B
JournalJ Biol Chem
PubMed ID1527036
Five antibiotics (puromycin, erythromycin, lincomycin, sparsomycin, and virginiamycin M1) that bind specifically to the 50 S ribosomal subunit near the peptidyl transferase center were used to compare and characterize the positions of bound AcylPhe-tRNA in the puromycin-reactive and -unreactive states. Binding of the antibiotics was quantitatively measured by their perturbation ... More