T. William Hutchens

Mapping and sequence-specific identification of phosphopeptides in unfractionated protein digest mixtures by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

We have demonstrated a procedure for the rapid (minutes), sensitive (

Protein interactions with immobilized transition metal ions: Quantitative evaluations of variations in affinity and binding capacity

The interaction of proteins with immobilized transition-metal ions proceeds via mechanisms influenced by metal type and degree of coordination, variations in mobile phase constituents, and protein surface architecture at or near the metal binding site(s). The contributions each of these variables make toward the affinity of protein surfaces for immobilized metal ions remain empirical. We have used equilibrium binding analyses to evaluate the influence of pH and competitive binding reagents on the apparent equilibrium dissociation constant (Kd) and binding capacity of immobilized Cu(II) and Ni(II) ions for several model proteins of known three-dimensional structure. Linear Scatchard plots suggested that 8/13 of the proteins evaluated interacted with immobilized metal ions via a single class of operational (Kd = 10-700 microM) binding sites. Those proteins with the highest affinities for the immobilized Cu(II) ions (5/13) showed evidence of multiple, non-identical or nonindependent binding sites. The effects of altered metal type, pH, and concentration of competitive affinity reagents (e.g., imidazole, free metal ions) on the apparent Kd and binding capacity varied in magnitude for individual proteins. The presence of free Cu(II) ions did not detectably alter either the affinity or binding capacity of the proteins for immobilized Cu(II) ions. The expected relationship between the relative chromatographic elution sequence and calculated affinity constants was not entirely evident by evaluation under only one set of conditions. Our results demonstrate the utility of nonchromatographic equilibrium binding analyses for the quantitative evaluation of experimental variables affecting the relative affinity and capacity of immobilized metal ions for proteins. This approach affords the opportunity to improve understanding and to vary the contribution of interaction mechanisms involved.

Estrogen Regulation of Lactoferrin Expression in Human Endometrium

PROBLEM: Lactoferrin is an iron‐binding glycoprotein that has been shown to be overexpressed in human endometrial carcinomas. The purpose of our present study is to investigate the possible role of estradiol in the expression of lactoferrin.

Occupancy of a C2-C2 type ‘Zinc-finger’ protein domain by copper Direct observation by electrospray ionization mass spectrometry☆

The metal ion specificity of most ‘zinc‐finger’ metal binding domains is unknown. The human estrogen receptor protein contains two different C2‐C2 type ‘zinc‐finger’ sequences within its DNA‐binding domain (ERDBD). Copper inhibits the function of this protein by mechanisms which remain unclear. We have used electrospray ionization mass spectrometry to evaluate directly the 71‐residue ERDBD (K180‐M250) in the absence and presence of Cu(II) ions. The ERDBD showed a high affinity for Cu and was completely occupied with 4 Cu bound; each Cu ion was evidently bound to only two ligand residues (net loss of only 2 Da per bound Cu). The Cu binding stoichiometry was confirmed by atomic absorption. These results (i) provide the first direct physical evidence for the ability of the estrogen receptor DNA‐binding domain to bind Cu and (ii) document a twofold difference in the Zn‐ and Cu‐binding capacity. Differences in the ERDBD domain structure with bound Zn and Cu are predicted. Given the relative intracellular contents of Zn and Cu, our findings demonstrate the need to investigate further the Cu occupancy of this and other zinc‐finger domains both in vitro and in vivo.

Recognition of transition metal ions by peptides Identification of specific metal-binding peptides in proteolytic digest maps by UV laser desorption time-of-flight mass spectrometry

Metal‐binding peptides in proteolytic digest maps have been identified by matrix‐assisted UV laser desorption time‐of‐flight mass spectrometry (LDTOF‐MS). The plasma and milk metal transport protein chosen to demonstrate this process, histidine‐rich glycoprotein (HRG), was purified and then digested with trypsin; the cleavage products were analyzed by LDTOF‐MS with dihydroxbenzoic acid as the matrix. The selective interaction of specific peptides with one or more Cu atoms was observed when Cu(II) ions were added to the digest mixture. At least one specific metal‐binding peptide was identified by computerized sequence analysis using the molecular mass data and available cDNA sequence. These results demonstrate the first direct observation by mass spectrometry of differential peptide—metal ion interactions in protein digest maps. The ability to evaluate peptide—metal ion interactions, including stoichiometry, with less than 1 pmol of sample improves significantly our ability to identify metal binding domains in metal‐binding proteins.

Rapid purification of porcine colostral whey lactoferrin by affinity chromatography on single-stranded DNA-agarose. Characterization, amino acid composition and N-terminal amino acid sequence

We have determined that the major iron-binding and DNA-binding protein in porcine colostral whey is lactoferrin. This lactoferrin was purified to homogeneity in one chromatographic step using immobilized single-stranded DNA-agarose. Although different in chromatographic behavior from human lactoferrin, the porcine lactoferrin purified in this manner was shown to be homogeneous by high-performance ion-exchange chromatography (Mono-S), immobilized metal ion (Cu2+) affinity chromatography, size-exclusion chromatography (TSK-4000SW), and reverse-phase (phenyl) chromatography. Electrophoresis on SDS-polyacrylamide gradient (10-20%) gels under reducing conditions showed the purified lactoferrin to be a single protein (silver-stained) of 78 kDa. Apolactoferrin purified in this manner bound iron and displayed a UV/VIS absorption spectrum indistinguishable from that of human lactoferrin. The molar absorption coefficient of hololactoferrin was 3.86 x 10(3) M-1 at 465 nm and 1.08 x 10(5) M-1 at 280 nm. Affinity elution analyses of the purified lactoferrin on immobilized DNA revealed that the affinity of this protein for DNA was independent of bound iron. Porcine lactoferrin was recognized by antibodies directed against human lactoferrin and bovine lactoferrin. The amino acid composition and N-terminal amino acid sequence analysis (30 residues) revealed a high degree of sequence homology with human, equine and bovine lactoferrin. These results demonstrate the effectiveness of immobilized DNA as a rapid and simple lactoferrin purification procedure and demonstrate the presence of a lactoferrin in porcine colostral whey with a high degree of sequence homology to human lactoferrin.

Metal ligand-induced alterations in the surface structures of lactoferrin and transferrin probed by interaction with immobilized copper(II) ions.

We have evaluated immobilized Cu(II) ions as a potential site-directed molecular probe to monitor ligand-induced alterations in protein surface structures. Metal ion-induced alterations in the surface structures of different lactoferrins (human and porcine), transferrins (human and rabbit), and ovotransferrin (chicken) were examined. Although these 78,000-dalton glycoproteins are related gene products with similar overall structure and function, they differ greatly in the number and distribution of surface-exposed electron-donor groups thought to interact with Cu(II) ions. Each of these proteins interacted with immobilized Cu(II) ions through sites which are distinct from the two specific high affinity metal binding sites identified for iron. In both the presence and absence of bound iron, transferrins interacted more strongly with the immobilized Cu(II) ions than did lactoferrins; ovotransferrin interacted only weakly. Although iron binding increased the affinities of lactoferrins for immobilized Cu(II), iron binding decreased the affinities of transferrins and ovotransferrin for immobilized Cu(II) ions. Iron-saturated and iron-free lactoferrins were resolved by pH gradient elution, but only in the presence of 3 M urea; they were not resolved by imidazole affinity elution. Conversely, the iron-saturated and iron-free forms of transferrin were only separated by imidazole affinity elution. Urea did not influence the resolution of apo and holo ovotransferrins by imidazole. The differential effects of urea and imidazole suggest the participation of different types of surface electron-donor groups. The progressive site-specific modification of surface-exposed histidyl residues by carboxyethylation revealed several lactoferrin forms of intermediate affinity for immobilized iminodiacetate-Cu(II) ions. In summary, independent of species, the affinity for immobilized Cu(II) ions increased as follows: iron-saturated ovotransferrin less than metal-free ovotransferrin less than apolactoferrin less than hololactoferrin much less than diferric or holotransferrin less than monoferric transferrin less than apotransferrin. We have demonstrated the use of immobilized Cu(II) ions to distinguish and to monitor ligand-induced alterations in protein surface structure. The results are discussed in relation to protein surface-exposed areas of electron-donor groups.

Formation of crystalloid inclusions in the small intestine of neonatal pigs: an immunocytochemical study using colloidal gold

SummaryEnterocytes of the small intestine in 1-day-old suckling piglets contain numerous vesicles in the apical cytoplasm and a large granule located beneath the nucleus. Within the next 3 days, these granules transform into electron-dense crystalloid inclusions. These membrane-bound inclusions are up to 10 μm in length and 1–2 μm in diameter, and they are composed of electron-dense lamellae 3.9 nm apart. Postembedding immunocytochemistry, using rabbit anti-porcine IgG and goat anti-rabbit IgG conjugated to 10 nm colloidal gold, revealed that both the granules and the crystalloid inclusions contained a high concentration of maternal IgG. Although the IgG content of the crystalloid inclusions was detected on epoxy-embedded sections, the use of LR White resin resulted in a much higher density of labelling. Quantification of the labelling density showed that the concentration of IgG in the crystalloid inclusions was approximately ten times higher than that in the lumenal colostrum and approximately three times higher than that in the granules. These observations showed that there are at least three compartments involved in the accretion of IgG in the small intestine of neonatal piglets: smaller apical endocytotic vesicles, large subnuclear granules and crystalloid inclusions. The role of these compartments in maternal immunoglobulin absorption and in the acquisition of passive immunity has yet to be explored.

Proposed Mechanisms for the Involvement of Lactoferrin in the Hydrolysis of Nucleic Acids

Lactoferrin has recently been proposed to have ribonuclease activity in the absence of bound iron. We and others have demonstrated previously that lactoferrin interacts with DNA and will bind a number of transition metal ions via surface-exposed histidyl residues. In the present study, we investigated the possibility that surface-bound copper ions on lactoferrin may catalyze the production of active oxygen species responsible for the hydrolysis of nucleic acids. Purified lactoferrin (apo- and holo-forms) was incubated with CuCl2 in solution to obtain lactoferrin with surface binding sites saturated by Cu(II)ions. the lactoferrin-Cu(II) complex was purified by Bio-Gel P-6 chromatography columns and tested for hydrolytic activity against DNA and RNA as analyzed by agarose gel electrophoresis. Incubation of lactoferrin-Cu(II) complexes with supercoiled plasmid Bluescript II SK DNA led to the rapid formation of relaxed open circular or linear forms of DNA characterized by changed electrophoretic mobility. Lactoferrin with bound Cu(II) also caused extensive degradation of yeast tRNA molecules in the presence of hydrogen peroxide. Covalent modification of surface-exposed histidyl residues by carboxyethylation with diethylpyrocarbonate abolished the lactoferrin-associated hydrolytic activity. These results indicate that lactoferrin-bound Cu(II) can indeed facilitate the hydrolysis of DNA and RNA molecules. Copper-binding sites on lactoferrin appear to serve as centers for repeated production of hydroxyl radicals via a Fenton-type Haber-Weiss reaction. Enhanced nuclease activity associated with elevated local concentrations of lactoferrin would promote microbial degradation.

Metal ion affinity adsorption of a Zn(II)-transport protein present in maternal plasma during lactation: structural characterization and identification as histidine-rich glycoprotein.

A high-affinity Zn(II)-binding protein has been purified to homogeneity (880-fold) from the plasma of lactating women by a single affinity adsorption step on columns of tris(carboxymethyl)ethylenediamine (TED)-agarose loaded with Zn(II) ions. Purity was evaluated by high-performance reverse-phase (phenyl) chromatography and by silver staining after SDS-polyacrylamide gradient gel electrophoresis. The mass of denatured Zn(II)-binding protein was estimated by SDS-polyacrylamide gradient gel electrophoresis to be 75 kDa under both reducing and nonreducing conditions; by matrix-assisted uv laser desorption time-of-flight mass spectrometry the purified protein mass was determined to be 66 kDa. The amino acid composition revealed a high content of His (13 mol%) and Pro (12 mol%). N-terminal amino acid sequence analysis (50 residues) identified the purified protein as histidine-rich glycoprotein (HRG). Immunoblots demonstrated the absence of fragments in the purified product. An enzyme-linked immunosorbent assay was developed; a 75% recovery of intact HRG from the immobilized Zn(II) ion affinity column was documented. The circular dichroism spectra for the purified human HRG in the far uv (260-178 nm) were similar to those published for human and rabbit serum HRG. These results demonstrate that TED-immobilized Zn(II) ions can be used as a new and efficient method for the isolation of structurally intact human plasma HRG.