Dennis W. Darnall

Selective recovery of gold and other metal ions from an algal biomass.

The authors observed that the pH dependence of the binding of Au/sup 3 +/, Ag/sup +/, and Hg/sup 2 +/ to the algae Chlorella vulgaris is different than the binding of other metal ions. Between pH 5 and 7, a variety of metal ions bind strongly to the cell surface. Most of these algal-bound metal ions can be selectively desorbed by lowering the pH to 2; however, Au/sup 3 +/, Hg/sup 2 +/, and Ag/sup +/ are all bound strongly at pH 2. Addition of a strong ligand at different pHs is required to elute these ions from the algal surface. Algal-bound gold and mercury can be selectively eluted by using mercaptoethanol. An elution scheme is demonstrated for the binding and selective recovery of Cu/sup 2 +/, Zn/sup 2 +/, Au/sup 3 +/, and Hg/sup 2 +/ from an equimolar mixture. 20 references, 2 figures.

Accumulation of elemental gold on the alga Chlorella vulgaris

Abstract The accumulation of Au(0) by lyophilized preparations of the alga Chlorella vulgaris has been investigated. Gold is bound to the algae upon suspending dried algal cells in solutions containing hydrogen tetrachloroaurate (III). Relative amounts of ionic and atomic algal-bound gold were determined by thiourea extraction. It was found that the amount of algal-bound atomic gold produced from ionic gold increased with time. The effect of algal-bound gold concentration on the rate and extent of gold reduction was observed. It is suggested that at least three different classes of sites are available for gold binding and reduction. The effect of Au(0) accumulation on the binding ability of gold-bound algae was also investigated, and an apparent enhancement of gold binding ability is reported.

The thermodynamics of bovine and porcine insulin and proinsulin association determined by concentration difference spectroscopy.

Difference spectroscopy was used to determine the equilibrium constants and thermodynamic parameters for the monomer-dimer association of bovine and porcine insulin and bovine proinsulin at pH 2.0 and 7.0. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -6.6 kcal/mol, -18 cal/mol-deg, and -12 kcal/mol, respectively. Porcine insulin behaved similarly to bovine insulin in its dimerization properties in that delta G degree 25, delta S degree, and delta H degree were found to be -6.8 kcal/mol, -14 cal/mol-deg, and -11 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine insulin were found to be -7.2 kcal/mol, -16 cal/mol/deg, and -12 kcal/mol, respectively. At pH 7.0 delta G degree 25, delta S degree, and delta H degree for dimerization of porcine insulin were -6.7 kcal/mol, -11.6 cal/mol-deg, and -10 kcal/mol, respectively. The similarity in the thermodynamic parameters of both insulin species at the different pHs suggests that there are minimal structural changes at the monomer-monomer contact site over this pH range. The dimerization of both insulin species is under enthalpic control. This may suggest that the formation of the insulin dimer is not driven by hydrophobic bonding but, rather, is driven by the formation between subunits of four hydrogen bonds in an apolar environment. At pH 2 delta G degree 25, delta S degree, and delta H degree for dimerization of bovine proinsulin were found to be -5.3 kcal/mol, -26 cal/mol-deg, and -13 kcal/mol, respectively. At pH 7 delta G degree 25, delta S degree, and delta H degree for dimerization of proinsulin were -5.9 kcal/mol, -4.2 cal/mol-deg, and -7.2 kcal/mol, respectively. Although the presence of the C-peptide on proinsulin does not drastically affect the overall free energy change of dimer formation (as compared to insulin), the other thermodynamic parameters are rather drastically altered. This may be because of electrostatic interactions of groups on the C-peptide with groups on the B-chain which are near the subunit contact site in the insulin dimer.

Distance measurements between the metal-binding sites in thermolysin using terbium ion as a fluorescent probe

Abstract A single terbium ion has been introduced into thermolysin replacing two of the four calcium ions, and the fluorescence properties of the protein-bound terbium have been studied. The fluorescence of Tb+3 is tremendously enhanced (∼7 × 103) upon binding and is significantly quenched when divalent cobalt is substituted for the zinc ion normally found in the enzyme. By use of the Forster equation for energy transfer the distance between the protein-bound Tb+3 and Co+2 in the active site was calculated to be 13.6±0.5 A. This agrees closely with the value of 13.9 A obtained from the crystal structure and suggests that energy transfer between the two metal ions bound to the protein takes place by a dipole-dipole mechanism.

Investigation of Eu(III) Binding Sites on Datura innoxia Using Eu(III) Luminescence

A pulsed tunable dye laser has been used to obtain excitation spectra and fluorescence decay curves of solid Eu(III)-Datura innoxia and from a series of Eu(III)-containing complexes. Carboxyl and sulfate groups have been demonstrated to be the dominant functional groups for forming binding sites on the cell wall of Datura innoxia at high (≥4) and low (≤3) pH conditions, respectively. The excitation spectra associated with the 7F0 → 5D0 electronic transition of Eu(III) luminescence have been used to provide a measure of the electronic structure factors contributing to the interaction between Eu(III) ions and the binding sites on the cell wall of D. innoxia. The noticeably broadened and asymmetric excitation spectra obtained at high pH conditions are ascribed to multiple binding sites. The corresponding lifetime decay curves exhibited a bi-exponential decay. A pK of 4.5 was determined for the binding of Eu(III) to the cell wall at pH ≥4. Kinetic and thermodynamic studies were also undertaken.

A study of carboxylic and amino acid complexes of neodymium(III) by difference absorption spectroscopy

Abstract The interaction of neodymium(III) with acetate, alanine, histidine, benzoate, and anthranilate has been studied using changes in the visible absorption spectrum of neodymium(III) upon complexation. At pHs below 6 only the carboxyl group of alanine coordinates to the metal ion, whereas at pHs near 7 the α-amino group begins to coordinate. The carboxyl group alone of histidine coordinates below pH 4. Between pH 4 and 6.5 the imidazole group of histidine coordinates forming a bidentate complex, and above pH 6.5 the α-amino group also begins to coordinate forming a tridentate complex. From a comparison of benzoate and anthranilate complexes it is clear that the amino group of anthranilate does coordinate to form a bidentate complex at a pH very near the pKa of the amino group of anthranilate.

The location of the lanthanide ion binding site on bovine trypsin

Summary Using the effect of a paramagnetic probe, Gd +3 , on the NMR relaxation time of inhibitor protons, the metal-inhibitor distances in a bovine trypsin ternary complex has been measured. The decrease due to Gd +3 in the spin-spin and spin-lattice relaxation times of the ortho and methyl protons of the inhibitor, p -toluamidine, has been measured at pH 6. The Solomon-Bloembergen equations were used to calculate distances of 8.8 ± 0.5 A and 10.0 ± 0.5 A from the metal ion to the ortho and methyl protons, respectively. From examination of the crystal structure of the enzyme it appears that the side chains of Asp 194 and Ser 190 are likely ligands for the metal ion.

The calcium ion binding site in bovine chymotrypsin A

Abstract The effect of Gd 3+ on the nuclear magnetic resonance (nmr) relaxation rates, T 1 m −1 and T 2 m −1 , of inhibitor protons in metal-inhibitor-α-chymotrypsin ternary complexes has been measured. The Solomon-Bloembergen equations were used to calculate the distance from the methyl protons of p -toluamidine (a competitive inhibitor) to the Gd 3+ binding site which is 9.2 ± 0.5 A. Calcium ion and gadolinium ion compete for the same binding site on α-chymotrypsin. Distances from the specificity pocket of α-chymotrypsin to the metal binding site have been measured by fluorescence energy transfer experiments. By observing energy transfer between proflavine and Nd 3+ , Pr 3+ , or Ho 3+ , we have been able to calculate a distance of approximately 10 A between the two chromophores. This agrees well with the data obtained by nmr techniques and also gives nearly identical values to those obtained for trypsin (Darnall, D., Abbott, F., Gomez, J. E., and Birnbaum, E. R., Biochemistry 15 , 5017, 1976). This is consistent with the calcium ion binding sites being composed of the same residues in both trypsin and α-chymotrypsin.

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.

The hydrolysis of 3-(2-furylacryloy)-glycyl-l-leucine amide by thermolysin

Abstract The kinetics of the hydrolysis of 3-(2-furylacryloyl)-glycycl- l -leucine amide by thermolysin has been reinvestigated. It was found that the K m for the enzyme substrate interaction is 2.5 × 10 −3 m at pH 7.2. This K m is an order of magnitude less than what has been previously assumed to be the K m for the enzyme-substrate interaction. The normally recommended assay has 1–3 × 10 −3 m substrate and is based on the assumption that the substrate concentration is much less than the K m . Our data indicate that this assumption appears to be invalid. The hydrolysis of 3-(2-furylacryloyl)-glycyl- l -leucine amide results in a maximum decrease in absorbance at 322 nm. The change in absorbance is nearly 10-fold greater at 322 nm than the change in absorbance at 345 nm where the hydrolysis has been customarily followed. By following the hydrolysis of the substrate at 10 −4 m at 322 nm it is possible to work under conditions where the substrate concentration is much less than the K m .