时间分辨荧光分析法在DNA探针检测中的初步应用

应用时间分辨荧光技术进行核酸杂交分析,选用自制整合剂异硫氰酸苯基-EDTA将铕离子标记连接于链霉亲和素分子中,通过光化学反应制备生物素标记pUC118DNA探针,与固定在聚苯乙烯微滴板中的靶DNA杂交后,以铕离子Eu(3+)标记的链霉亲和素为检测物,检测靶DNA的含量,可检测到30pg的靶DNA．     

Mechanism of non-enzymic transamination reaction between histidine and α-oxoglutaric acid

Non-enzymic transamination reactions at 85 degrees between various amino acids and alpha-oxoglutaric acid are catalysed by metal ions, e.g. Al(3+), Fe(2+), Cu(2+) and Fe(3+). The reaction is optimum at pH4.0. Of the 14 amino acids studied histidine is the most active. In the presence of Al(3+) histidine transaminates with alpha-oxoglutaric acid, forming glutamic acid and Al(3+)-imidazolylpyruvic acid complex as the end products. However, in the presence of Fe(2+) or Cu(2+) the products are glutamic acid and a 1:2 metal ion-imidazolylpyruvic acid chelate. The greater effectiveness of histidine in these reactions is attributed to the presence of the tertiary imidazole nitrogen atom, which is involved in the formation of stable sparingly soluble metal ion-imidazolylpyruvic acid complexes or chelates as end products of these reactions. Of the metal ions studied only Al(3+), Fe(2+), Fe(3+) and Cu(2+) are effective catalysts for the transamination reactions, and EDTA addition completely inhibits the catalytic effect of the Al(3+). Spectrophotometric evidence is presented to demonstrate the presence of metal ion complexes of Schiff bases of histidine as intermediates in the transamination reactions. These results may contribute to understanding the role of histidine in enzyme catalysis.     

Synthesis and fluorescence properties of the europium(III) chelate of a polyacid derivative of terpyridine.

A new polyacid derivative ligand of biphenyl-substituted terpyridine, [4'-(biphenyl-4'-yl)-2,2':6'2'-terpyridine-6,6'-diyl]bis(methylenenitrilo)tetrakis(acetic acid) was synthesized, and the fluorescence properties of its Eu3+ and Tb3+ chelates were investigated. The Eu3+ chelate of the ligand is strongly fluorescent, with a fluorescence quantum yield of 0.156, molar absorption coefficient of 2.95 x 10(4) mol/L/cm at molar absorption maximum of 336 nm, and fluorescence lifetime of 1.29 ms, whereas its Tb3+ chelate is non-fluorescent.     

Closed-tube human leukocyte antigen DQA1∗05 genotyping assay based on switchable lanthanide luminescence probes

Genotyping in closed tube is commonly performed using polymerase chain reaction (PCR) amplification and allele-specific oligonucleotide probes using fluorescence resonance energy transfer (FRET). Here we introduce a homogeneous human leukocyte antigen (HLA)–DQA1∗05 end-point PCR assay based on switchable lanthanide luminescence probe technology and a simple dried blood sample preparation. The switchable probe technology is based on two non-luminescent oligonucleotide probes: one carrying a non-luminescent lanthanide chelate and the other carrying a light-absorbing antenna ligand. Hybridization of the probes in adjacent positions to the target DNA leads to the formation of a highly luminescent lanthanide chelate complex by self-assembly of the reporter molecules. Performance of the HLA–DQA1∗05 assay was evaluated by testing blood samples collected on sample collection cards and was prepared by lysing the punched samples (3-mm discs) using alkaline reaction conditions and high temperature. Testing of 147 blood samples yielded 100% correlation to the heterogeneous DELFIA technology-based reference assay. Genotyping requires carefully designed probe sequences able to discriminate matched and mismatched target sequences by hybridization. Furthermore, definite genotype discrimination was achieved because inherently non-luminescent switchable probes together with time-resolved measurement mode led to very low background signal level and, therefore, very high signal differences averaging 54-fold between DQA1∗05 and other alleles.     

Observation of calcium uptake by isolated sarcoplasmic reticulum employing a fluorescent chelate probe

The fluorescent chelate probe technique is employed to observe the accumulation and binding of Ca⁺⁺ to isolated sarcoplasmic reticulum from skeletal and cardiac muscle. Chlorotetracycline serves as a fluorescent chelate probe which chelates to membrane bound Ca⁺⁺ giving rise to an intensely fluorescence adduct. An increase in fluorescence of chlorotetracycline is caused by ATP induced Ca⁺⁺ transport in both skeletal and cardiac muscle microsomes. The fluorescence spectra indicate that Ca⁺⁺ lies on the membrane surface in a relatively polar environment.     

Nonradioactive detection of nucleic acid by the universal probe system

A convenient and nonradioactive method for DNA hybridization tests termed the "Universal probe system" has been developed. This method is based on the principle of sandwich hybridization. This system consists of two single-stranded DNA probes (a primary probe and a biotin-labeled secondary probe). The primary probe is prepared from a chimeric phage-plasmid vector containing the complementary sequence to a target gene. The secondary probe has a sequence complementary to the vector portion of the primary probe and is labeled with biotin via the transamination reaction. An advantage of this method is that the single-stranded primary probe can be prepared with ease by using the chimeric phage-plasmid vector system, thereby avoiding tedious labeling of individually different probes. As the primary probe is not modified with biotin and other labels, it conserves the sequence to be hybridized with a target. Accordingly, the primary probe containing a relatively short hybridizing region (ca. 50 bp) can efficiently hybridize with the target. In fact, the universal probe is sensitive enough to detect a single-copy human gene on Southern blots.     

A novel bifunctional europium chelate applied in quantitative determination of human immunoglobin G using time-resolved fluoroimmunoassay

The authors demonstrate herein a novel time-resolved fluoroimmunoassay (TRFIA) protocol for quantification of human IgG with the new bifunctional chelate Eu(TTA)₃(5-NH₂-phen) (ETNP) labeling the goat anti-human IgG. The immunoassay was conducted by following the typical procedure for sandwich-type immunoreactions. Goat anti-human IgG was immobilized on aldehyde-modified glass slides. The human IgG analyte was first captured by the primary antibody and then sandwiched by a secondary antibody labeled with the chelate ETNP. The experimental procedure was simple to follow and gave desirable levels of sensitivity and low limits of detection. To the best of our knowledge, this is the first application of the new chelate, ETNP, in an immunoassay. In comparison to typical organic, fluorescent compounds and other lanthanide fluorescent chelates used in immunoassay, the detection sensitivity of our method using ETNP chelate in the solid phase was greatly improved and a concentration of human IgG about 5 μg/L could be detected under optimal conditions. The main result of this work shows that the new chelate ETNP can be applied as a powerful fluorescent labeling material for constructing ultrasensitive TRFIAs. The detection of human IgG, using ETNP as the chelate, is a model example of the effectiveness of this immunoassay. Many other types of antigen–antibody immunoassays should be possible using the protocol described herein.     

Labelling of histone H5 and its interaction with DNA. 2. Cooperative binding of histone H5 to DNA as probed by steady-state fluorescence and diffusion-enhanced energy transfer

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors. Analysis of the binding data by the model of Schwarz and Watanabe (J.Mol.Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 microM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range. DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A and TbEDTA- chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

Metal affinity precipitation of proteins

Proteins containing multiple surface-accessible histidine residues can be precipitated using small quantities of bis-copper chelates. The chelates serve to crosslink the proteins, presumably via the accessible histidines, leading to the formation of large, insoluble complexes. When excess copper chelate is used to carry out the precipitation, the resulting precipitate has a stoichiometry of 1:1 copper:accessible histidine. The precipitation is analogous to antibody-antigen precipitin reactions and can be described qualitatively using simple equilibrium theory developed for those systems. Human hemoglobin contains a large number of surface histidines and is efficiently precipitated by the copper salt CuSO4 as well as by bis-copper chelates. Sperm whale myoglobin contains many fewer surface histidines and is precipitated only by the bis-chelates. The effects of the number of accessible histidines on the protein, the chain length separating the two chelates, and the pH on the precipitation reaction have been investigated.     

Comparison of a tartaric acid derived polymeric MRI contrast agent to a small molecule model chelate

Contrast agents with high relaxivity are needed to increase the sensitivity of magnetic resonance imaging (MRI) for novel clinical and research applications. For this reason, polymeric structures containing multiple Gd(III) chelates are of current interest. Described in this communication are the syntheses and characterization of a glycopolymer derived from L-tartaric acid, Gd 4(H2O), as well as a low molecular weight compound, Gd 10(H2O), that models the Gd(III) chelate structure in the repeat unit of polymer Gd 4(H2O). Luminescence lifetime measurements in H2O and D2O for Eu(III) analogues of Gd 4(H2O) and Gd 10(H2O) [named Eu 4(H2O) and Eu 10(H2O)] reveal that the lanthanide in both structures likely has one water ligand in the primary coordination sphere. The relaxivity of the model chelate Gd 10(H 2O) at 400 MHz and 310 K was determined to be 4.7 mmol (-1).s (-1), representing a nearly 50% increase over Magnevist (3.2 mmol (-1).s (-1)). Relaxivity values on a per Gd basis for the polymeric structure Gd 4(H2O) prepared at two degrees of polymerization, n = 12 and 19, are similar, but slightly lower than Gd 10(H2O) (4.4 mmol (-1).s (-1) and 4.5 mmol (-1).s (-1), respectively). However, their molecular relaxivities of 51 mmol (-1).s (-1) and 80 mmol (-1).s (-1), respectively, provide a substantial increase over that of Magnevist.     

Construction of DNA-probes and their immunochemical detection using nonradioactive hybridization tests

Simple and efficient chemical approaches to preparation of DNA probes carrying 2,4-dinitrophenyl, dansyl or biotin residues were developed. The residues were introduced using following DNA derivatization procedures: a) transamination of cytidine residues with O-(4-aminobutyl)hydroxylamine; b) mercuration of pyrimidine residues followed by beta-mercaptoethanol modification. It was shown that 2,4-dinitrophenyl-containing DNA probes can be used for nonradioactive hybridization detection of nucleic acids. DNP-DNA: DNA complexes were detected using mouse antibodies specific to 2,4-dinitrophenyl groups, which were developed with peroxidase-conjugated antimouse immunoglobulins. Peroxidase-catalyzed chemoluminescent reaction of luminol oxidation with hydrogen peroxide allowed to detect 10 picograms of the dinitrophenylated single-stranded DNA probe.     

Carbostyril derivatives as antenna molecules for luminescent lanthanide chelates

Luminescent lanthanide complexes consisting of a lanthanide-binding chelate and organic-based antenna molecule have unusual emission properties, including millisecond excited state lifetimes and sharply spiked spectra, compared to standard organic fluorophores. We have previously used carbostyril (cs124, 7-amino-4-methyl-2(1H)-quinolinone) as an antenna molecule (Li and Selvin, J. Am. Chem. Soc., 1995) attached to a polyaminocarboxylate chelate such as DTPA. Here, we report the chelate syntheses of DTPA conjugated with cs124 derivatives substituted on the 1-, 3-, 4-, 5-, 6-, and 8-position. Among them, the DTPA chelate of cs124-6-SO(3)H has similar lifetime and brightness for both Tb(3+) and Eu(3+) compared to the corresponding DTPA-cs124 complexes, yet it is significantly more soluble in water. The Tb(3+) complex of DTPA-cs124-8-CH(3) has significantly longer lifetime compared to DTPA-cs124 (1.74 vs 1.5 ms), indicating higher lanthanide quantum yield resulting from the elimination of back emission energy transfer from Tb(3+) to the antenna molecule. Thiol-reactive forms of chelates were made for coupling to proteins. These lanthanide complexes are anticipated to be useful in a variety of fluorescence-based bioassays.     

Diffusion-enhanced lanthanide energy-transfer study of DNA-bound cobalt(III) bleomycins: comparisons of accessibility and electrostatic potential with DNA complexes of ethidium and acridine orange

Energy transfer in the "rapid-diffusion" limit reflects the equilibrium properties of a donor-acceptor system. Rates of energy transfer from freely diffusing terbium chelates to DNA-binding chromophores change dramatically when DNA is added; energy transfer from an electrically neutral chelate is reduced because the energy acceptor becomes partially buried in DNA, while energy transfer from a positive chelate is increased because of electrostatic attraction. The rate constants for energy transfer to DNA-bound chromophores from a positively charged terbium chelate, relative to those from a neutral chelate, were used to estimate the following values for the electrostatic potential near the surface of each DNA-bound acceptor at 298 K in the presence of 1.0 mM added salt (in units of -e/kT): acridine orange, 4.54 +/- 0.11; ethidium, 4.66 +/- 0.07; green Co(III) bleomycin A2, 4.06 +/- 0.11; orange Co(III) bleomycin A2, 3.11 +/- 0.10. Smaller numbers indicate less negative potentials; these can be due to a combination of (1) positive charge on the chromophore, (2) location of the chromophore [particularly Co(III) bleomycin] away from the DNA phosphates, and/or (3) separation of DNA phosphate negative charges by an intercalator. The magnitudes of the individual rate constants indicate that all the DNA-bound chromophores can be directly encountered by the terbium probes. Energy-transfer rate constants from a neutral terbium chelate to DNA-bound and free acceptors can provide a measure of the accessibility of the terbium probe to each bound chromophore. The ratios of these rate constants were as follows: acridine orange, 0.17 +/- 0.01; ethidium, 0.27 +/- 0.02; green form of Co(III) bleomycin A2, 0.48 +/- 0.06; orange form of Co(III) bleomycin A2, 0.71 +/- 0.06. These results are consistent with the probable differences in binding mechanisms for the intercalating chromophores (ethidium and acridine orange) as compared to the Co(III) bleomycins (in which the relevant chromophores are nonintercalating metal centers). In addition, all the results imply that the green Co(III) bleomycin chromophore binds closer to DNA than the orange; this provides a first step toward understanding the structural basis for the different biological properties of these metallobleomycins. Control experiments and theoretical considerations necessary to establish the validity of the results are also presented.     

A homogeneous DNA hybridization system by using a new luminescence terbium chelate

Homogeneous DNA hybridization assay based on the luminescence resonance energy transfer (LRET) from a new luminescence terbium chelate, N,N,N(1),N(1)-[2,6-bis(3'-aminomethyl-1'-pyrazolyl)-4-phenylpyridine]tetrakis(acetic acid) (BPTA)-Tb(3+) (lambda(ex) = 325 nm and lambda(em) = 545 nm) to an organic dye, Cy3 (lambda(ex) = 548 nm and lambda(em) = 565 nm), has been developed. In the system, two DNA probes whose sequences are complementary to the two different consecutive sequences of a target DNA are used; one of the probes is labeled with the Tb(3+) chelate at the 3'-end, and the other is with Cy3 at the 5'-end. Labeling of the Tb(3+) chelate is accomplished via the linkage of a biotin-labeled DNA probe with the Tb(3+) chelate-labeled streptavidin. Strong sensitized emission of Cy3 was observed upon excitation of the Tb(3+) chelate at 325 nm, when the two probe DNAs were hybridized with the target DNA. The sensitivity of the assay was very high compared with those of the previous homogeneous-format assays using the conventional organic dyes; the detection limit of the present assay is about 30 pM of the target DNA strand.     

Spectrophotometric determination and removal of unchelated europium ions from solutions containing Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates

Europium chelates conjugated with peptide ligands are routinely used as probes for conducting in vitro binding experiments. The presence of unchelated Eu ions in these formulations gives high background luminescence and can lead to poor results in binding assays. In our experience, the reported methods for purification of these probes do not achieve adequate removal of unchelated metal ions in a reliable manner. In this work, a xylenol orange-based assay for the quantification of unchelated metal ions was streamlined and used to determine levels of metal ion contamination as well as the success of metal ion removal on attempted purification. We compared the use of Empore chelating disks and Chelex 100 resin for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate–peptide conjugates. Both purification methods gave complete and selective removal of the contaminant metal ions. However, Empore chelating disks were found to give much higher recoveries of the probes under the conditions used. Related to the issue of probe recovery, we also describe a significantly more efficient method for the synthesis of one such probe using Rink amide AM resin in place of Tentagel S resin.     

The characterisation of structural and antioxidant properties of isoflavone metal chelates

Isoflavone metal chelates are of interest as isoflavones act as oestrogen mimics. Metal interactions may enhance isoflavones biological properties so understanding isoflavone metal chelation is important for the commercial application of isoflavones. This work aimed to determine if isoflavones, daidzein (4',7-dihydroxyisoflavone) and genistein (4',5,7-trihydroxyisoflavone) could chelate with metals as isoflavone chelates. Biochanin A (4'-methoxy-5,7-dihydroxyisoflavone) was also examined for it's ability to chelate with Cu(II) and Fe(III). This study found daidzein does not chelate with Cu(II) and Fe(III) but genistein and biochanin A chelate with a 1:2 M/L stoichiometry. The copper and iron chelates were synthesised and characterised by elemental analysis, FTIR, thermogravimetric analysis (TGA) and electrospray ionisation mass spectrometry (ESI-MS). These studies indicated a 1:2 M/L stoichiometry and suggested the isoflavones bind with the metals at the 4-keto and the 5-OH site. 2,2-diphenyl-1-picrylhydrazyl (DPPH) inhibition assays showed that copper isoflavone chelates have higher antioxidant activity than free isoflavones while the iron isoflavone chelates showed pro-oxidant activity compared to the free isoflavone. Synergistic DPPH studies with 0.02 mM ascorbic acid revealed copper chelates exhibit reduced antioxidant activity versus free isoflavones whereas the iron chelates showed lower pro-oxidant activity except at 1.0 mM.     

Index to Subjects,Volume 5

Abstract

The interaction of histone H5 labelled with fluorescein isothiocyanate (FITC) with DNA has been studied by fluorescence titration, and diffusion-enhanced fluorescence energy transfer (DEFET) measurements with Tb(III) lanthanide chelates as donors.

Analysis of the binding data by the model of Schwarz and Watanabe (J. Mol. Biol. 163, 467-484 (1983)) yielded a mean stoichiometry of 60 nucleotides per H5 molecule, independently of ionic strength, in the range of 3 to 300 mM NaCl, at very low DNA concentration (6 μM in mononucleotide). It ensues an approximate electroneutrality of the saturated complexes. Histone H5 molecules appeared to be clustered along the DNA lattice in clusters containing on average 3 to 4 H5 molecules separated by about 79 base pairs, at mid-saturation of the binding sites. The interaction process was found highly cooperative but the cooperativity parameter was also insensitive to ionic strength in the above range.

DEFET experiments indicated an important decrease of accessibility of the FITC label to the TbHED3A° and TbEDTA^? chelates with ionic strength in the 0 to 100 mM NaCl range. In the presence of DNA, H5 appears already folded at low ionic strength so that the FITC probe is also not accessible to the donor chelate. The present study constitutes an indispensable preliminary step to further studies on the localization of histone H5 in condensed chromatin structures.     

Formation and structure of reaction products of cis-PtCl2(NH3)2 with d(ApG) and/or d(GpA) in di-, tri- and penta-nucleotides. Preference for GpA chelation over ApG chelation

The reaction products of cis-PtCl2(NH)3)2 with several deoxyribonucleotides containing d(ApG) and/or d(GpA) have been studied. The various reaction products were separated by high-performance liquid chromatography and characterized by means of absorbance at 254 nm in combination with atomic absorption spectroscopy and 300-MHz 1H-NMR (pH dependence of the non-exchangeable base-protons, T1 relaxation time determinations). For the larger fragments the results from these techniques were confirmed by enzymatic degradation studies of the platinated fragments. The smallest of the investigated nucleotides, d(ApG) and d(GpA), both formed a variety of different platinum chelates. In the reaction with d(ApG) 15% cis-Pt(NH3)2-[d(ApG)N1(1),N7(2)] and 78% cis-Pt(NH3)2[d(ApG)N7(1),N7(2)] were found, 4% of the reacted material consisted of a 1 mol Pt/2 mol dinucleotide product, and 3% of an unidentified 1:1 product. From the main product two rotamers were found to occur: at room temperature, 81% anti,anti and 19% anti,syn product is present. With d(GpA) about equal amounts of N1,N7 and N7,N7 products were found; for both products the anti,anti and anti,syn conformations were found, respectively. Upon reaction of cis-PtCl2(NH3)2 with d(pApG) and d(pGpA) only the N7,N7 products were found; at room temperature and pH greater than 1.5 these products were present in anti,anti conformation. However, for the d(pApG)-platinum chelate at -20 degrees C a small amount (less than 5%) of a second product could be observed in NMR. For the d(pGpA)-platinum chelate a second N7,N7-coordinated product was observed when the pH of the NMR sample was lowered to 1.1 (at this pH the free 5'-phosphate group is protonated). With the larger fragments d(ApGpA), d(pApGpA) and d(TpApGpApT) the intra-molecular competition between the formation of the d(ApG) or the d(GpA) chelates could be studied. Using these nucleotides no N1-coordinated products or rotamers were observed. In the case of d(ApGpA) and d(TpApGpApT) the d(GpA) chelate (67% and 75% respectively) was favoured over the d(ApG) chelate, while with d(pApGpA) about equal amounts of both chelates were formed.     

Purification and immunochemical studies of pyruvate dehydrogenase complex from rat heart, and cell-free synthesis of lipoamide dehydrogenase, a component of the complex

A mixture of NADPH and ferredoxin reductase is a convenient way of reducing adriamycin in vitro. Under aerobic conditions the adriamycin semiquinone reacts rapidly with O2 and superoxide radical is produced. Superoxide generated either by adriamycin:ferredoxin reductase or by hypoxanthine:xanthine oxidase can promote the formation of hydroxyl radicals in the presence of soluble iron chelates. Hydroxyl radicals produced by a hypoxanthine:xanthine oxidase system in the presence of an iron chelate cause extensive fragmentation in double-stranded DNA. Protection is offered by catalase, superoxide dismutase or desferrioxamine. Addition of double-stranded DNA to a mixture of adriamycin, ferredoxin reductase, NADPH and iron chelate inhibits formation of both superoxide and hydroxyl radicals. This is not due to direct inhibition of ferredoxin reductase and single-stranded DNA has a much weaker inhibitory effect. It is concluded that adriamycin intercalated into DNA cannot be reduced.     

Application of a new surface labeling reagent, EDTA derivative, on erythrocytes and platelets.

The modes of binding of a new class of impermeant metal-chelating probe, the complex of 111In3+ to 1-(p-benzenediazonium) ethylenediamine tetraacetic acid (azo-phenyl-EDTA), to human and rabbit erythrocyte membranes and the effect of binding on the function of rabbit platelets have been studied. The metal chelate, azo-phenyl-EDTA.[111In3+] bound covalently to membrane proteins following reaction with intact erythrocytes. The amount and the pattern of labeling was assessed by sodium dodecyl sulfate (SDS)-polyacrylamide disc and slab gels for radioactivity. The pattern of labeling of intact human erythrocytes by azo-phenyl-EDTA.[111In3+], by pyridoxal phosphate-NaB3H7 and by galactose oxidase-NaB3H4 was also compared. The following results were obtained: (a) The pattern of labeling of intact human erythrocyte by azo-phenyl-EDTA.[111In3+] differed from other commonly used probes for labeling external membrane surfaces. Five polypeptides were labeled by the metal chelates. In addition to the known major proteins (protein band III, PAS-1, PAS-2 and PAS-3 of Fairbanks et al. (1972) Biochemistry 10, 2606--2617) a protein (radioactive band 4) which migrated slightly slower than PAS-3 in SDS gel was labeled heavily by the metal chelate. This protein has an apparent molecular weight of 37,500 in 8.4% acrylamide-SDS gel. About 40% of bound radioactivity was found in this protein. The diazo linkage of the metal chelate to this protein was found to be especially unstable to heat. (b) In rabbit erythrocyte membranes, the metal chelate bound to three polypeptides with apparent molecular weights of 96,000, 43,000 and 33,000 in 8.4% acrylamide gel. They are probably glycoproteins in nature. (c) The binding of the probe to platelets did not affect the platelet aggregability induced by adenosine diphoshpate. In vivo studies indicated that the labeled platelets accumulated at the plague of atherosclerotic rabbits. (d) The bifunctional analog of EDTA may permit new applications of metals with useful physical properties for studies of cell membranes.