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E R Birnbaum

Publications and source records attributed to E R Birnbaum.

At least 19 recordsLinked to original sources
PubMed1990

Eu3+ luminescence studies of oncomodulin. The origin of the pH-dependent behavior.

The Eu3+ 7F0----5D0 excitation spectra of parvalbumin and oncomodulin are pH-dependent. Until now, it had been assumed that both the CD and EF ion-binding sites shared this property and that deprotonation of water molecules coordinated to the bound Eu3+ ions might be responsible for the pH dependence. Results obtained with the site-specific variant of oncomodulin known as D59E, in which glutamate replaces the aspartate naturally present at position 59, have necessitated substantial revision of these ideas. It now appears that the pH-dependent behavior is confined to the CD site. Moreover, we observe no corresponding change in the number of O-H oscillators coordinated to the bound Eu3+ ions in the pH range over which we observe the spectroscopic alteration. It is likely that the behavior results from deprotonation of one or more carboxyl groups clustered at the COOH-terminal end of the CD domain.

Amino Acid Sequence↗
PubMed1989

Site-specific substitution of glutamate for aspartate at position 59 of rat oncomodulin.

Replacement of the aspartate residue at position 59 of rat oncomodulin by glutamate by oligonucleotide-directed mutagenesis has afforded a protein which more closely resembles rat parvalbumin, at least judged by its interaction with the luminescent lanthanide ion Eu3+. The single-peak 7F0----5D0 spectrum observed at pH 5.0 with the fully bound wild-type protein is replaced by one which clearly shows two features at 5791 and 5796 A, arising from Eu3+ ions bound at the CD and EF sites, respectively. Furthermore, the pH dependence of the spectrum is substantially altered; the pKa observed for the CD domain, in which aspartate 59 residues, is shifted upward from pH 6.0 for the wild-type recombinant protein to pH 6.8 in the D59E mutant. Moreover, the maximum in the high-pH spectrum is shifted from 5781 to 5784 A. All three changes are indicative of a CD binding domain having increased parvalbumin-like character. Interestingly, however, the D59E substitution has only a modest effect on the Ca2+- and Mg2+-binding properties of the CD domain. For the wild-type protein, KCa = 7.8 x 10(-7) M and KMg = 3 x 10(-3) M. These affinities are more than an order of magnitude weaker than those seen for various parvalbumins and substantiate previous claims for calcium specificity made for the oncomodulin CD domain. Replacement of aspartate 59 by glutamate resulted in minor increases in affinity of the CD domain for Ca2+ (KCa = 5.5 x 10(-7) M) and Mg2+ (KMg = 1 x 10(-3) M). These findings strongly suggest that residues in oncomodulin besides aspartate 59 are important determinants of the observed calcium specificity of the CD calcium-binding domain. The consequences of the substitution at residue 59 appear to be confined to the CD domain. For the EF site in wild-type recombinant oncomodulin, KCa = 4.2 x 10(-8) M and KMg = 1.6 x 10(-4) M. The corresponding values for the D59E site-specific variant are identical within experimental error (KCa = 4.2 x 10(-8) M and KMg = 1.8 x 10(-4) M).

Animals↗
PubMed1988

Oncomodulin and parvalbumin. A comparison of their interactions with europium ion.

The 7F0----5D0 transition of Eu3+ was used to probe the metal-binding domains of rat oncomodulin and rat parvalbumin. Two distinct differences between the two proteins were observed. The first relates to the pH-dependent behavior of their 7F0----5D0 spectra, a phenomenon noted previously for other paravalbumins. In the case of rat parvalbumin, the spectral features associated with both metal-binding sites titrate concomitantly (pK alpha = 8.2); however, in the case of oncomodulin, the two sites titrate sequentially (pK alpha = 6.3 for the CD site; pK alpha = 8.3 for EF site). The proteins also contrast with regard to their discrimination for Eu3+ over Ca2+. The CD and EF sites in rat parvalbumin both display a large preference for Eu3+: (KCa/KEu)CD = 143 +/- 11 and (KCa/KEu)EF = 191 +/- 30. However, in the case of oncomodulin, although the EF site of oncomodulin greatly prefers the trivalent lanthanide ion (KCa/KEu = 300 +/- 80), the CD site exhibits a relatively minor preference (KCa/KEu = 11 +/- 1).

Animals↗
PubMed1986

Lanthanide-binding properties of rat oncomodulin.

Oncomodulin, the parvalbumin-like calcium-binding protein frequently expressed in tumor tissue, was isolated from Morris hepatoma 5123tc and studied using the luminescent lanthanide ions, Eu3+ and Tb3+. Titrations of the apoprotein - whether monitored by indirect excitation of bound Tb3+, by direct laser excitation of bound Eu3+, or by quenching of the intrinsic tyrosine fluorescence - all indicated the presence of two high-affinity binding sites for lanthanide ions, as in parvalbumin. Moreover, the appearance of the Eu3+ 7F0----5D0 excitation spectrum of Eu2-oncomodulin was found to be highly pH-dependent, as previously observed with parvalbumin. At pH 5.0, it consists of a single peak centered at 5796 A, having a linewidth of approximately 6 A. At higher pH values, this spectrum is replaced by a broader, more symmetric peak at 5782 A. Oncomodulin, however, was found to differ from parvalbumin in at least one important respect: In contrast to the muscle-associated protein, the affinities of the CD site in oncomodulation for Tb3+ and Ca2+ were found to be rather similar, with KCa/KTb approximately equal to 11 +/- 2.

Animals↗
PubMed1985

Luminescence studies of lanthanide ion binding to parvalbumin.

Luminescence methods were used to examine the interaction of Eu(III) and Tb(III) with parvalbumin isozyme III from pike (Esox lucius). The bound lanthanide ions were excited both directly, via laser irradiation, and indirectly, via fluorescence energy transfer from adjacent phenylalanine residues. At high (175 microM) protein concentrations, the lanthanide titration curves exhibited pronounced quenching of luminescence at Ln3+:parvalbumin ratios above 2:1, in agreement with earlier reports (Donato, H., Jr., and Martin, R. B. (1974) Biochemistry 13, 4575-4579). However, in experiments performed with lower concentrations (10 microM), the titrations were well behaved and indicated a lanthanide:protein stoichiometry of 2:1. Equilibrium dialysis measurements performed with Eu(III) ruled out the existence of a third strong binding site which could cause the quenching of the luminescence at high protein concentrations. Similarly, careful analysis of the spectrum that results from direct excitation of the 7F0----5D0 transition of parvalbumin-bound Eu3+ ion revealed no peak attributable to a third Ln3+-binding site. The peak which has been construed by others (Rhee, M.-J., Sudnick, D. R., Arkle, V. K., and Horrocks, W. DeW., Jr. (1981) Biochemistry 20, 3328-3334) as evidence for a third site was shown to result from a pH-dependent spectral transition involving the europium ions bound at the CD and EF sites. Luminescent lifetime measurements performed on Tb(III)/parvalbumin solutions follow Stern-Volmer quenching kinetics at terbium:protein ratios in excess of 2:1, suggesting that the quenching results from collisional deactivation of the tightly bound ions by excess terbium ion free in solution.

Animals↗
PubMed1984

Anthraniloyl-tyrosine 411 as a spectroscopic probe of fatty acid binding to human serum albumin.

Reaction of human serum albumin with p-nitrophenylanthranilate results in transesterification of the anthraniloyl group to tyrosine 411. Titration of anthraniloyl-Tyr-411-albumin with long chain or short chain fatty acids produces marked changes in the absorption and fluorescence spectra of the anthraniloyl moiety as fatty acids bind in the channel near it. It appears that the anthraniloyl group is a very sensitive probe that can follow binding of small molecules at the 3-AB subdomain of human serum albumin.

Fatty Acids↗
PubMed1984

Synthesis of a new chelating gel: removal of Ca2+ ions from parvalbumin.

The synthesis of a chelating gel which contains the effective metal chelating agent ethylenediaminetetraacetic acid covalently linked to amino-agarose is described. This gel is shown to be a rapid and extremely effective material for the removal of tightly bound, but labile metal ions from proteins without introducing contaminants into the biological system. The synthesis involves the formation of an amide linkage between the dangling carboxylate arm of the [Co(EDTA)Cl]2-complex and amino-agarose using a standard carbodiimide coupling reaction. The chelating gel is shown to remove approximately 98.5% of the calcium from fully bound Ca2-parvalbumin and over 99% of the europium from Eu2-parvalbumin.

Calcium↗
PubMed1984

Synthesis of affinity-label chelates: a novel synthetic method of coupling ethylenediaminetetraacetic acid to amine functional groups.

An affinity-label chelate for the enzyme trypsin was synthesized by a novel synthetic technique which takes advantage of the presence of a dangling carboxylate arm in the [Co(EDTA)Cl]2- complex anion. The dangling carboxylate group was coupled to the amino group of p-aminobenzamidine, an effective inhibitor of trypsin activity, via the carbodiimmide reaction to produce a trypsin affinity label at one end and a strong EDTA-like chelating agent at the other, coupled through an amide bond. The cobalt ion can be removed if desired by reduction with Fe2+ + ascorbate, and alternate metal ions inserted in its place. The reaction is general, and affinity labels which contain amino groups can be easily coupled via this procedure, allowing the introduction of a paramagnetic or fluorescent probe into a protein or nucleotide system. The same method has been used to prepare a highly effective chelating gel which is capable of removing calcium and lanthanide ions from the binding protein parvalbumin.

Affinity Labels↗
PubMed1983

Resonance energy transfer between cysteine-34 and tryptophan-214 in human serum albumin. Distance measurements as a function of pH.

The single cysteine residue (Cys-34) of human serum albumin was modified with the organic mercurial [4-[p-(dimethylamino)phenyl]azo]phenyl]mercuric acetate. Introduction of this chromophore into the protein results in the quenching of the protein tryptophan fluorescence spectrum due to energy transfer from the tryptophan residue to the mercurial. Since human albumin contains only a single tryptophan, it was then possible to calculate distances between the mercurial bound at Cys-34 and Trp-214 under various conditions. This distance contracted during the course of the N leads to F transition, being 34-35 A in the N conformation (pH 6-7.5) and 29.9 A in the F conformation (pH 3.6). The distance increased substantially during the course of the F leads to E transition occurring between pH 3.6 and pH 1.9 and was found to be nearly 37 A at pH 1.9. The distance between Cys-34 and Trp-214 was found to undergo a slight contraction during the N leads to B transition occurring between pH 7.0 and pH 9.0. At pH 8.5-9 where the protein is predominately in the B form, the distance was found to be slightly more than 31 A.

Cysteine↗
PubMed1983

Resonance energy transfer between cysteine-34, tryptophan-214, and tyrosine-411 of human serum albumin.

Reaction of p-nitrophenyl anthranilate with human serum albumin at pH 8.0 results in esterification of a single anthraniloyl moiety with the hydroxyl group of tyrosine-411. The absorption spectrum of the anthraniloyl group overlaps the fluorescence emission of the single tryptophan residue at position 214. This study complements that of the preceding paper [Suzukida, M., Le, H. P., Shahid, F., McPherson, R. A., Birnbaum, E.R., & Darnall, D. W. (1983) Biochemistry (preceding paper in this issue)] where an azomercurial group was introduced at cysteine-34. Anthraniloyl fluorescence was also quenched by the azomercurial absorption at Cys-34. Thus measurement of resonance energy transfer between these three sites allowed distances to be measured between Cys-34 in domain I, Trp-214 in domain II, and Tyr-411 in domain III of human serum albumin. At pH 7.4 in 0.1 M phosphate the Trp-214 leads to Tyr-411, Tyr-411 leads to Cys-34, and Trp-214 leads to Cys-34 distances were found to be 25.2 +/- 0.6, 25.2 +/- 2.1, and 31.8 +/- 0.8 A, respectively.

Circular Dichroism↗
PubMed1982

The lanthanide-induced N = to F transition and acid expansion of serum albumin.

Serum albumin exists in the native or N conformation between pH 5 and 7. As the pH is lowered from 5 to 3.5, the protein undergoes a conformational change resulting in expansion, known as the N = to F (partially acid expanded) transition. As the pH is lowered still further to 2, the protein continues to expand. In the present study, using the techniques of circular dichroism, fluorescence, and UV difference spectroscopy, lanthanide ions at concentrations between 1-30 mM have been shown to produce both changes in the albumin structure analogous to the N = to F transition and acid expansion of bovine serum albumin at a constant pH near 6.

Cations↗
PubMed1980

Tyrosine fluorescence as a measure of denaturation in thermolysin.

The heat and guanidine hydrochloride denaturation of thermolysin has been followed by fluorescence techniques. The native enzyme has a single emission peak which is decreased in intensity and which splits into two clearly resolved peaks upon denaturation. These data are interpreted to indicate that energy transfer from tyrosine to tryptophan occurs in the native enzyme which is lost upon denaturation. Even though zinc is fully bound to thermolysin at 90 degrees C or in the presence of 6 M guanidine hydrochloride, removal of zinc from the denatured enzyme has no effect on the emission spectrum. Removal of Ca2+ from the denatured enzyme. These data indicate that even though the metal ions are bound to the denatured protein, they provide little structural integrity to the protein as measured by energy transfer between tyrosine and tryptophan.

Energy Transfer↗
PubMed1977

The effect of calcium ion on the urea denaturation of immobilized bovine trypsin.

The effect of calcium ion on the urea denaturation of trypsin has been investigated. By using trypsin immobilized on glass beads, all possibilities of autolysis occurring during the denaturation process are eliminated. It was found that in 8 M urea calcium ion markedly decreases the denaturation rate of the immobilized trypsin. Conversely, the presence of calcium ion markedly accelerates the rate of renaturation of denatured immobilized trypsin. Calcium may exert its stabilizing effect on the tertiary structure of the protein by coordination to the side chains of Asp 194, Ser 190 and the carbonyl group of Ser 139 (using the chymotryptic numbering system).

Animals↗
PubMed1976

Fluorescence energy-transfer measurements between the calcium binding site and the specificity pocket of bovine trypsin using lanthanide probes.

Using fluorescence energy-transfer experiments we have measured the distance between the specificity pocket and the calcium ion binding site of bovine pancreatic trypsin. Proflavin and thionine were used to block the specificity site, whereas various lanthanide ions were substituted for the calcium. It was then possible to choose various donor-acceptor pairs which exhibit suitable energy transfer. We have calculated the distance between proflavin and Nd(III), Pr(III), and Ho(III) to be 10.9, and 10.3, and 10.3 A, respectively. This agrees very well with the value of approximately 10 A we obtained between the methyl protons of p-toluamidine (a competitive inhibitor) and Gd(III) using nuclear magnetic resonance techniques (Abbott, F., Gomez, J.E., Birnbaum, E.R., and Darnall, D.W. (1975), Biochemistry 14, 4935). This is strong evidence that, in solution, the calcium binding site is composed of the side chains of Ser-190 and Asp-194.

Amino Acid Sequence↗