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Two isoforms of a protease inhibitor were isolated by ion-exchange chromatography of tepary bean (Phaseolus acutifolius G.) seed proteins. The main isoform was used to determine the amino acid sequence of the protein. It is an 80 amino acid residue protein with a molecular mass of 8765 Da, showing sequence homology with the Bowman-Birk family of protease inhibitors. Several regions with amino acid microheterogeneity were found, corroborating the possible presence of isoforms. Mass spectrometry analysis was carried out to confirm isoforms. The presence of dimer and trimer forms of the inhibitor was shown through electrophoresis and mass spectrometry. Another unusual characteristic for this inhibitor was its ability to bind metals. The presence of four sequential histidines at the N-terminal end of the protein could account for this binding. Mass spectrometry and atomic absorption spectroscopy support the presence of calcium in the native inhibitor.

The ability to define specific alterations in the structure and function of proteins as they are introduced and processed in vivo remains an important goal. We have evaluated the generation, in vivo, of an antimicrobial peptide (lactoferricin) derived from ingested bovine lactoferrin by surface-enhanced laser desorption/ionization (SELDI). SELDI was used in the affinity mass spectrometry operational mode to detect and quantify lactoferricin directly from unfractionated gastric contents using a chemically defined ligand with a terminal n-butyl group as the lactoferricin affinity capture device. By this method, we were able to detect and quantify lactoferricin directly upon examination of unfractionated gastric contents recovered from an adult subject 10 min after ingestion of bovine lactoferrin (200 ml of 10 mg/ml (1.2 x 10(-4) mol/l) solution). Lactoferricin produced in vivo was directly captured by a surface-enhanced affinity capture (SEAC) device composed of molecules with a terminal n-butyl group and analyzed by laser desorption/ionization time-of-flight mass spectrometry. The recovery of standard lactoferricin or lactoferrin added to an aliquot of the gastric contents was determined to be nearly 100%, confirming the efficiency of this method. The amount of lactoferricin detected in the gastric contents was 16.9+/-2.7 microg/ml (5.4+/-0.8 x 10(-6) mol/l). However, a large proportion of ingested lactoferrin was found to be incompletely hydrolyzed. Lactoferrin fragments containing the lactoferricin region were analyzed by in situ pepsin hydrolysis after being captured on the SEAC device. Partially degraded lactoferrin fragments containing the lactoferricin region, including fragments corresponding to positions 17-43, 17-44, 12-44, 9-58 and 16-79 of the bovine lactoferrin sequence, were found to be present at concentrations as high as 5.7+/-0.7 x 10(-5) mol/l. These results suggest that significant amounts of bovine lactoferricin would be produced in the human stomach following ingestion of food, such as infant formula, supplemented with bovine lactoferrin. We propose that physiologically functional quantities of human lactoferricin could be generated in the stomach of breast-fed infants, and possibly, in the case of adults, from lactoferrin secreted into saliva.

Lactoferrin is an 80 kDa major protein component of mammalian colostral whey. The antimicrobial active centre of lactoferrin, lactoferricin (Lfcin), may also be an important determinant of the interaction between lactoferrin and specific receptors on lymphocytes. We have documented the survival in vivo of ingested lactoferrin in the gastrointestinal tract of adult mice by surface-enhanced laser desorption/ionization affinity MS. Various kinds of degraded lactoferrin fragments were detected as molecular-ion peaks corresponding to Lfcin after being captured by an affinity capture device, hydrolysis in situ and laser desorption/ionization. No evident molecular-ion peaks of Lfcin were observed upon analysis of faeces from mice fed commercial milk, whereas lactoferrin fragments containing the Lfcin region were detected at concentrations in the order of at least pmol/g in the faeces of mice fed milk enriched with lactoferrin at 40 mg/ml. These results suggest that ingested lactoferrin would survive transit through the gastrointestinal tract as partially degraded forms containing the receptor-binding region(s) as well as the antimicrobial active centre.

Lactoferricin is a bioactive peptide fragment (3196 Da) derived from lactoferrin (80 kDa) that contains the bactericidal domain and the lymphocyte receptor-binding domain of lactoferrin. Although lactoferricin has been produced from lactoferrin by proteolytic digestion in vitro, its natural occurrence and distribution in vivo are still not clear, in part because of the absence of a suitable detection means. Surface-enhanced laser desorption/ionization (SELDI) was used to detect and characterize lactoferricin by affinity mass spectrometry. Human, porcine, and bovine lactoferricin in unfractionated serum samples were found to bind specifically to ligands presenting a terminal n-butyl group. SELDI was used to detect and quantify each species of lactoferricin in a manner that was independent of the presence of intact lactoferrin, partially degraded lactoferrin, and lactoferrin peptides containing the lactoferricin peptide sequence. The limit of detection of bovine lactofericin in serum was as low as 200 pg/ml. The FKCRRWQWR-homoserine/-homoserine lactone moiety of bovine lactoferricin, which includes the complete antimicrobial center (i.e., RRWQWR), was shown to be responsible for interaction with the n-butyl group. The SELDI procedure defined here is the only molecular recognition tool known to date that is capable of distinguishing the multi-functional lactoferricin domain located within structurally related but distinct forms of lactoferrin and its metabolic fragments. Enabling the direct quantitation of lactoferricin produced in vivo opens new opportunities to evaluate lactoferrin function.

Lactoferricin (Lfcin) is a bioactive fragment of lactoferrin derived from the bactericidal and putative lymphocyte receptor binding domain(s) located within the N-lobe of lactoferrin. Although known to be liberated from at least three species of lactoferrin, conditions leading to Lfcin generation in vivo and factors affecting its distribution are still not known. Recently, we have developed a method of surface-enhanced laser desorption/ionization (SELDI) affinity mass spectrometry using n-butyl terminal groups for surface-enhanced affinity capture (SEAC) to quantify not only Lfcin generated in vivo but also other lactoferrin fragments. Unlike previous efforts to detect lactoferrin and Lfcin with specific antibodies, the SELDI affinity assay distinguished lactoferrin, lactoferrin fragments, Lfcin and unrelated peptides without their interference with each other. To evaluate Lfcin generation in vivo, the experimental design involved feeding 200 mL of 10 mg/mL (1.22 x 10(-4) mol/L) bovine lactoferrin to an adult. Gastric contents were recovered 10 min after ingestion. Lfcin produced in vivo was directly captured by the SEAC device. The amount of Lfcin in the gastric contents was 16.91 +/- 2.65 micrograms/mL (5.350 +/- 0.838 x 10(-6) mol/L). However, a large proportion of the ingested lactoferrin was not completely digested. Lactoferrin fragments containing the Lfcin region were analyzed by in situ hydrolysis with pepsin after being captured by the SEAC device. As much as 5.740 +/- 0.702 x 10(-5) mol/L of the partially degraded lactoferrin fragments were found to contain the Lfcin region, including peptide domains 17-43, 17-44, 12-44, 9-58, and 16-76 of bovine lactoferrin. These results show that bovine Lfcin can be produced in the human stomach after ingestion of an infant formula supplemented with bovine lactoferrin. It is now important to determine whether Lfcin is generated in the intestinal tract of formula-fed and breast-fed infants, and geriatric patients consuming foods enriched with lactoferrin.

The human progesterone receptor (hPR) in T47D breast cancer cells is phosphorylated on at least nine different serine residues. We have previously reported the identification of five sites; three are hormone inducible (Ser102, Ser294 and Ser345), and their phosphorylation correlates with the timing of the change in receptor mobility on gel electrophoresis in response to hormone treatment. The other two sites, Ser81 and Ser162, along with the remaining sites, are basally phosphorylated and exhibit a general increase in phosphorylation in response to hormone. With the exception of Ser81, all of these sites are in Ser-Pro motifs, suggesting that proline-directed kinases are responsible for their phosphorylation. We now report that cyclin A-cyclin-dependent kinase-2 complexes phosphorylate hPR-B in vitro with a high stoichiometry on three sites that are authentic basal sites in vivo. One of these is Ser162, which has been described previously. The other two sites are identified here as Ser190 and Ser400. The specificity and stoichiometry of the in vitro phosphorylation suggest that hPR phosphorylation may be regulated in a cell cycle-dependent manner in vivo.

Affinity mass spectrometry (AMS) was used to evaluate the structural diversity of the E2 component of pyruvate dehydrogenase complex (PDC) in normal and diseased liver cells, including those from patients with the autoimmune disease primary biliary cirrhosis (PBC). Two different antibodies to PDC-E2, the immunodominant mitochondrial autoantigen in patients with PBC, were used. AMS was performed directly on frozen liver sections and purified bile duct epithelial cells. Mass spectrometric signals associated with the molecular recognition of PBC-specific antigenic determinants were enhanced by an in situ enzyme-linked signal amplification process. Samples from patients with PBC gave strong positive signals for the antigen(s) recognized by the monoclonal antibody C355.1. Conversely, tissues from normal and disease controls showed only a minimal signal. AMS was used to identify specific antigenic determinants within the E2 component of PDC for comparison with unknown antigenic determinants observed by affinity capture with C355.1 monoclonal antibody from PBC samples. PDC components bound to C355.1 were mapped and identified by mass before dissociation from the E2 component. A similar approach was used to identify unknown antigenic determinants associated with PBC. We believe AMS may be an important new approach with wide application to the identification of molecules associated with a number of disease states.

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., Method of Study: We investigated 1) serum levels of lactoferrin in five women during normal ovulatory cycles, 2) serum levels of lactoferrin during ten human menopausal gonadotropin induced cycles when estradiol levels are high, and 3) lactoferrin expression in five proliferative and five secretory phase endometrium by immunohistochemical studies. The serum concentrations of lactoferrin were measured by a peroxidase-based enzyme-linked immunosorbent assay., Results: In normal ovulatory cycles, the mean serum lactoferrin concentration during the proliferative phase (0.4013 +/- 0.0242 micrograms/mL) was significantly higher (P < 0.02) than in the secretory phase (0.3468 +/- 0.0209 micrograms/mL). In induced cycles, there was gradual increase in lactoferrin levels with increasing estradiol concentrations. Peak lactoferrin levels in induced cycles (0.7495 +/- 0.1148 micrograms/mL) were significantly higher (P < 0.003) than the midcycle levels (0.423 +/- 0.0424 micrograms/mL) in normal cycles. Immunohistochemical analysis of the endometrium revealed greater expression of lactoferrin in proliferative endometrium (50.7 +/- 13%, range 28-72%) than in secretory endometrium (19.2 +/- 4%, range 7-31%)., Conclusion: These results indicate that estradiol may play a role in the regulation of lactoferrin expression in human endometrium.

Quantitative aspects of oligonucleotide analysis by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry remain largely unexplored relative to the efforts that have been devoted to quantitative peptide and protein analysis. The successful quantitation of these other biopolymers coupled with the potential of rapid nucleic acid analysis by desorption/ionization techniques prompted the present investigation into quantifying mixed base oligonucleotides of intermediate molecular weights. This report describes the concentration-dependent desorption/ionization of a 21-base oligonucleotide (MW 6361) using a 36-base oligonucleotide (MW 11 131) as an internal standard. Peak height and peak area ratios (analyte to internal standard) varied linearly as a function of oligonucleotide concentration (R2 = 0.966 and 0.991, respectively). The linearity of response extended over nearly three orders of magnitude, from 0.125 to 100 pmol of analyte applied. The use of an internal standard improved the linearity of the calibration curve and reduced relative standard deviations. These results demonstrate for the first time the quantitation of medium size oligonucleotides using MALDI.

Lactoferrin, an iron-binding glycoprotein that is abundant in milk of some species, has been suggested to play a key role in the absorption of iron in human infants. This hypothesis is based on the dominant role of lactoferrin as an iron-binding component in human milk and on the occurrence of lactoferrin receptors in brush-border membranes in infants' intestines. The piglet may be a useful model to evaluate the biological activity of lactoferrin because we have previously found the presence of a lactoferrin receptor in brush-border membranes from suckling piglets. In this study, viable enterocytes were isolated from 6- to 20-day-old suckling piglets. Binding studies were performed at 4 degrees C using 125I-labeled porcine lactoferrin. Scatchard analysis of equilibrium binding data showed an apparent binding constant (Kd) of 2 x 10(-6) M (SD = 0.6 x 10(-6)). This affinity is in close agreement with previous results obtained using isolated brush-border membrane vesicles. Bovine lactoferrin inhibited the binding of porcine lactoferrin. Porcine transferrin, however, did not affect porcine lactoferrin binding significantly. Thus, lactoferrin binding is highly specific. When enterocytes were incubated with 125I-labeled lactoferrin at 37 degrees C, the amount of cell-associated radioactivity exceeded the surface binding capacity of the cells by almost fivefold. This finding agrees with the continuous binding and subsequent internalization of 125I-labeled lactoferrin. The isolated piglet enterocyte seems to provide a useful model for further studies of the mechanism of receptor-mediated absorption of lactoferrin.

This article describes the technique of immobilized metal ion affinity chromatography (IMAC). The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups in peptides and on protein surfaces. The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there is enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. The versatility of IMAC is one of its greatest assets. An important contribution to the correct use of IMAC for protein purification is a simplified presentation of the various sample elution procedures.

Intact (i.e., 78-kDa) lactoferrin has been purified from the urine of preterm infants fed human milk. The maternal origin of this lactoferrin, and the integrity of its primary structure have been documented. Computer analyses of the circular dichroism spectra revealed a composite secondary structure for the urinary lactoferrin that was indistinguishable from that of purified human milk lactoferrin and similar to that observed in the crystal structure. Intact function was suggested by iron binding; an approximate 2:1 molar ratio of iron to lactoferrin was confirmed. Thus, maternal lactoferrin is absorbed intact by the preterm infant and appears to remain structurally and functionally intact within the circulatory system and during urinary excretion. It is possible, therefore, that maternal lactoferrin has an immunoregulatory influence in newborn infants fed human milk.

Specific receptors for lactoferrin (Lf) have been identified in the small intestine brush-border membrane and on lymphocytes. Little is known about the mechanism of Lf interaction with its receptor protein. Because of the pH dependence of this interaction, the possible involvement of surface exposed histidine residues was explored. His residues on purified human milk Lf were progressively modified by carboxyethylation with diethyl pyrocarbonate; the reaction was monitored by UV absorbance and by affinity chromatography on immobilized Cu(II) ions. Human infant brush-border membrane vesicles (BBMVs) were prepared by differential centrifugation and precipitation methods. Human peripheral lymphocytes were prepared by Ficoll centrifugation and stimulated by mitogen (PHA) exposure in culture. A competitive receptor binding assay was performed with 59Fe- and/or 125I-labelled native Lf and His-modified Lf. Binding assays with labelled native Lf were conducted in the presence of a 10-200 fold molar excess of DEPC-Lf, the DEPC-Lf was not competitive. A 100-fold molar excess of native Lf completely blocked the interaction of labelled Lf with the receptor. Similar results were obtained for receptor proteins on BBMVs and lymphocytes. Thus, Lf receptors on these two different cell types appear to require His residues for Lf binding.

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.

We estimated in vivo turnover rates of sucrase-isomaltase and lactase-phlorizin hydrolase in adult rats. Fed animals received a primed continuous infusion of phenylalanine (300 microCi, 150 mumol Phe/100 g of body weight for 30 s, then 7.5 microCi, 3.75 mumol Phe/min for 10 to 140 min). Sucrase-isomaltase and lactase-phlorizin hydrolase were immunoprecipitated from jejunal mucosal membranes; isoforms were separated by SDS-polyacrylamide gel electrophoresis. Endoglycosidase H digestions and (for lactase-phlorizin hydrolase) N-terminal amino acid sequencing were performed on all isoforms. Specific radioactivity of prosucrase-isomaltase and prolactase-phlorizin hydrolase isoforms reached isotopic equilibrium by 60 and 90 min, respectively. Specific radioactivity of brush border sucrase and lactase did not reach steady state. The isotope kinetic, N-terminal amino acid sequencing, and endoglycosidase H digestion data suggested that one of the high molecular weight lactase isoforms is a dimer of mature lactase. Compartmental modeling of specific radioactivity demonstrated that mean intracellular residence time is 59 min for prosucrase-isomaltase isoforms and 68 min for prolactase-phlorizin hydrolase isoforms. Mean residence time in the brush border was 5.8 h for sucrase and 7.8 h for lactase. Fractional synthesis rates were 414%/day for sucrase and 307%/day for lactase. Thus, in vivo brush border sucrase and lactase turn over at similar rates in the adult rat.

Enterocytes 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 microns in length and 1-2 microns 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.

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.

We have demonstrated a procedure for the rapid (minutes), sensitive (less than pmol), and sequence-specific identification of phosphopeptides in unfractionated digests of phosphoproteins using matrix-assisted UV laser desorption/ionization (MALDI) time-of-flight (TOF) mass spectrometry. The mass-dependent identification of one specific 13-residue phosphopeptide (S105-K117), observed among the 153 possible trypsin digest fragments of human beta-casein (211 residues), was confirmed by amino acid sequence analysis of the 33P-labeled peptide after isolation by reverse-phase HPLC. MALDI-TOF was also used to monitor the rate and extent to which an 18-residue N-terminal beta-casein peptide (R1-K18) was phosphorylated in vitro. These results demonstrate that MALDI-TOF may be used (i) to facilitate the identification of sequence-specific sites of protein phosphorylation and dephosphorylation, (ii) to monitor protein and peptide phosphorylation and dephosphorylation reaction rates, even in complex unfractionated mixtures, (iii) to determine the minimum primary structure necessary for the phosphorylation of specific protein surface domains, and (iv) to evaluate the effects of intact protein phosphorylation and dephosphorylation on susceptibility to subsequent proteolytic events.

Transition metal ions are important in biological regulation partly because they can bind to and stabilize protein surface domain structures in specific conformations that are involved in key molecular recognition events. There are two C2-C2 type zinc-finger sequences within the highly conserved DNA-binding domain of the estrogen receptor protein (ERDBD). Electrospray ionization (ESI) mass spectrometry has been used to demonstrate that the metal-binding sites within the 71-residue ERDBD can bind either Zn (up to 2) or Cu (up to 4). Evidence for the induction and/or stabilization of a different conformational state with bound Cu is revealed by a characteristic shift in the ESI charge envelope. The 10+ charge state is most abundant for the fully reduced ERDBD apopeptide and the ERDBD-Zn holopeptide (bound Zn does not alter the charge envelope). In contrast, the 8+ charge state is typically the optimum charge state observed for the ERDBD-Cu holopeptide; indeed, the entire charge envelope is frame-shifted to lower charge states with bound Cu. Interpretation of the altered charge states is simplified because (i) a single type of metal-binding ligand (sulfur) is involved in the case of both Zn and Cu binding, and (ii) the two different metal cations are both divalent. Thus, it is likely that the dissimilar charge envelopes represent different peptide conformers, each of which is stabilized by a different type of bound metal ion.(ABSTRACT TRUNCATED AT 250 WORDS)

This preliminary investigation tests the premise that biologically relevant (1) peptide-metal ion interactions, and (2) metal ion-dependent macromolecular recognition events (e.g., peptide-peptide interactions) may be modeled by biomimetic affinity chromatography. Divinylsulfone-activated agarose (6%) was used to immobilize three different synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein histidine-rich glycoprotein (HRG). The synthetic peptides represented 1-3 multiple repeat units of the 5-residue sequence (Gly-His-His-Pro-His) found in the C-terminal of HRG. By frontal analyses, immobilized HRG peptides of the type (GHHPH)nG, where n = 1-3, were each found to have a similar binding capacity for both Cu(II) ions and Zn(II) ions (31-38 mumol/ml gel). The metal ion-dependent interaction of a variety of model peptides with each of the immobilized HRG peptide affinity columns demonstrated differences in selectivity despite the similar internal sequence homology and metal ion binding capacity. The immobilized 11-residue HRG peptide was loaded with Cu(II) ions and used to demonstrate selective adsorption and isolation of proteins from human plasma. These results suggest that immobilized metal-binding peptides selected from known solvent-exposed protein surface metal-binding domains may be useful model systems to evaluate the specificity of biologically relevant metal ion-dependent interaction and transfer events in vitro.

To extend the analytical capabilities of immobilized metal ion affinity chromatography (IMAC) for evaluation of biologically relevant peptide-metal ion interactions, we have prepared synthetic peptides representing metal-binding protein surface domains from the human plasma metal transport protein known as histidine-rich glycoprotein (HRG). Three synthetic peptides, representing multiples of a 5-residue repeat sequence (Gly-His-His-Pro-His) from within the histidine- and proline-rich region of the C-terminal domain were prepared. Prior to immobilization, the synthetic peptides were evaluated for identity and sample homogeneity by matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), a method developed recently for the mass determination of high-molecular-mass biopolymers. 2,5-Dihydroxybenzoic acid was evaluated as a matrix to facilitate the laser desorption and ionization of intact peptides and was found to be ideally suited for determinations of mass within the low-mass region of interest (641.7 to 1772.8 dalton). We observed minimal chemical noise from photochemically generated peptide-matrix adduct signals, clustering, and multiply-charged peptide species. Peptides with bound sodium and potassium ions were observed; however, these signal intensities were reduced by immersion of the sample probe tip in water. Mixtures of the three different synthetic peptides were also evaluated by LDTOF-MS after their elution through a special immobilized peptide-metal ion column designed to investigate metal ion transfer. We found LDTOF-MS to be a useful new method to verify the presence of peptide-bound metal ions.(ABSTRACT TRUNCATED AT 250 WORDS)

Conditions have been developed to characterize the reversible interaction of one or more Zn(II) ions with cysteine (sulfur) ligands on metal-binding peptides by electrospray-ionization (ES) mass spectrometry. A 71-residue peptide with two separate clusters of four cysteine residues was selected as a model to optimize both the solution and electrospray variables most likely to affect the detection of stable cysteine (sulfur) ligand/Zn interactions. By infusing peptide in water alone, stable electrospray and ion signals were produced in both the absence and presence of up to 100 microM zinc sulfate. In the absence of Zn(II), the calculated mass of the fully reduced peptide (8248.5 Da) was observed (8248.4 +/- 0.4 Da). In the presence of Zn(II), peptides with zero, one and two bound Zn atoms were detected; all three species were present in several different charge states. The overall charge envelope was typically unchanged in the presence of Zn; the charge-state optimum (10+) observed for this peptide was apparently unaffected by the presence of bound Zn. The interaction of Zn(II) ions with sulfur ligands in this peptide appeared to result in tetracoordinate covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Histidine-rich glycoprotein (HRG) is a 74-kD glycoprotein, originally discovered in plasma, which contains an unusually large amount of histidine (13 mol%) and proline (13 mol%). The specific functions of this protein remain unclear, although it binds (reversibly) transition metal ions such as Cu(II) and Zn(II) with high capacity (10-13 equivalent) and moderate to high affinity (kd = 0.2-10 microM). Because the bioavailability of Cu(II) and Zn(II) ions in human milk is high, we have used specific antibodies from polyclonal antisera directed against purified human plasma HRG to investigate whether this or a related protein is a component of human colostrum and(or) mature milk. Fresh human colostrum (d 1-3) and milk (d 4-120) were collected in the presence and absence of multiple protease inhibitors and EDTA. Immuno "dot" blot analyses and ELISA were developed; HRG was present in both colostrum (0.13-10 micrograms/mL) and mature milk (0.1-10 micrograms/mL). Unidentified components in colostrum and milk, however, were found to depress HRG antigenicity in these assays. Western transfer and immunoblots of denatured colostrum and milk samples analyzed by SDS-PAGE revealed the presence of an immunoreactive band at 74-78 kD, with other bands at 47 and 24 kD under both reducing and nonreducing conditions; smaller immunoreactive fragments (12-14 kD) were detected in some samples. We observed at least one additional band of immunoreactivity of greater molecular mass (greater than 110 kD) in colostrum under nonreducing conditions; we did not observe these bands in plasma samples. Immunoaffinity and Zn(II) affinity isolation of HRG from colostrum and milk resulted in the copurification of several associated proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

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 dihydroxybenzoic 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.

Thiophilic adsorption is useful for the purification of immunoglobulins under mild conditions (e.g., see ref. 1). Although there are several established procedures for the purification of immunoglobulins (2-5), thiophilic adsorption appears thus far to be unique in its capacity to adsorb three major classes of immunoglobulins (and their subclasses) (6-8). Furthermore, in contrast to other affinity purification methods (e.g., see refs. 3,4), recovery of the adsorbed (purified) immunoglobulins from the thiophilic adsorption matrix is accomplished efficiently at neutral pH, without the need for perturbation of protein structure (1). The most important utility of thiophilic adsorption is perhaps its use for the selective depletion of immunoglobulins from complex biological fluids (e.g., calf serum and hybridoma culture media, colostrum and milk) (6,7,9). This latter development has been particularly useful with hybridoma cell culture applications (9,10), and in the investigation of milk-immunoglobulin function during early periods of human infant nutrition (6,7).

Immobilized metal ion affinity chromatography (IMAC) (1,2) is also referred to as metal chelate chromatography, metal ion interaction chromatography, and ligand-exchange chromatography. We view this affinity separation technique as an intermediate between highly specific, high-affinity bioaffinity separation methods, and wider spectrum, low-specificity adsorption methods, such as ion exchange. The IMAC stationary phases are designed to chelate certain metal ions that have selectivity for specific groups (e.g., His residues) in peptides (e.g., 3-7) and on protein surfaces (8-13). The number of stationary phases that can be synthesized for efficient chelation of metal ions is unlimited, but the critical consideration is that there must be enough exposure of the metal ion to interact with the proteins, preferably in a biospecific manner. Several examples are presented in Fig. 1. The challenge to produce new immobilized chelating groups, including protein surface metal-binding domains (14,15) is being explored continuously. Table 1 presents a list of published procedures for the synthesis and use of stationary phases with immobilized chelating groups. This is by no means exhaustive, and is intended only to give an idea of the scope and versatility of IMAC. Fig. 1 Schematic illustration of several types of immobilized metal-chelating groups, including, iminodiacetate (IDA), tris(carboxymethyl) ethylenediamine (TED), and the metal-binding peptides (GHHPH)(n)G (where n = 1,2,3, and 5) (14,15). Table 1 Immobilized Chelating Groups and Metal Ions Used for Immobilized Metal Ion Affinity Chromatography Chelating group Suitable metal ions Reference Commercial source Immodiacetate Transitional1,2 Pharmacia LKB Pierce Sigma Boehringer Mannheim TosoHaas 2-Hydroxy-3[N-(2- pyrtdylmethyl) glycme]propyl Transitional3 Not available ?-Alky1 mtrilo triacetic acid Transitional4 Not available Carboxymethylated asparhc acid Ca(II)13 Not available Tris (carboxy- methyl) ethylene Diamme Transitional2 Not available (GHHPH)(n)G* Transitional14,15 Not available *Letters represent standard l-letter amino acid codes (C = glycine, H = histidine, P = proline) The number of internal repeat units is given by n(n= 1, 2, 3, and 5).

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.

A relatively recent method developed to determine the molecular weights of intact peptides and proteins, matrix-assisted UV laser desorption time-of-flight mass spectrometry (LDTOF-MS), has been evaluated as a new means to investigate the metal ion-binding properties of model synthetic peptides. A contiguous sequence of 25 residues on the surface of the 74 kDa human plasma metal-binding transport protein histidine-rich glycoprotein (HRG) has been identified as a bioactive metal-binding domain. The peptide, (GHHPH)5G, was synthesized and evaluated by LDTOF-MS before and after the addition of Cu(II) in solution with 2,5-dihydroxybenzoic acid as the matrix. In the absence of added Cu(II), the major protonated molecular ion (M + H)+ was observed to have a mass equal to its calculated mass (2904.0 Da). In the presence of Cu(II), however, five additional peaks were observed at mass increments of approximately 63.9 Da. The maximum Cu(II)-binding capacity observed for the 26-residue peptide (5 g-atoms/mol) suggested that up to 1 Cu(II) may be bound per 5-residue internal repeat unit (GHHPH) within this peptide; several other monovalent and divalent metal cations were not bound under identical conditions of analysis. The Cu(II)-binding stoichiometry was verified by spectrophotometric titration and by frontal analyses of the immobilized peptide with a solution of Cu(II) ions. These results demonstrate the ability to verify directly the solution-phase binding capacity of metal-binding peptides by LDTOF-MS.

We have used equilibrium binding analyses to evaluate the influence of temperature and urea on the affinity of hen egg white lysozyme and bovine pancreatic ribonuclease A for surface-immobilized Cu(II) ions. Linear Scatchard plots suggested that these model proteins were interacting with immobilized metal ions via a single class of intermediate-affinity (Kd = 10-40 microM) binding sites. Alterations in temperature had little or no effect on the immobilized Cu(II) binding capacity of either protein. Temperature effects on the interaction affinity, however, were protein-dependent and varied considerably. The affinity of lysozyme for immobilized Cu(II) ions was significantly decreased with increased temperature (0 degree C-37 degrees C), yet the affinity of ribonuclease did not vary measurably over the same temperature range. The van 't Hoff plot (1n K vs 1/T) for lysozyme suggests a straight line relationship (single mechanism) with a delta H of approximately -5.5 kcal/mol. Urea effects also varied in a protein-dependent manner. A 10-fold reduction in the affinity of lysozyme for the immobilized Cu(II) was observed with the urea concentrations up to 3 M; yet urea had no effect on the affinity of ribonuclease for the immobilized metal ions. Although the interaction capacity of lysozyme with the immobilized Cu(II) ions was decreased by 50% in 3 M urea, ribonuclease interaction capacity was not diminished in urea. Thus, temperature- and urea-dependent alterations in protein-metal ion interactions were observed for lysozyme but not ribonuclease A. The complete, yet reversible, inhibition of lysozyme- and ribonuclease-metal ion interactions by carboxyethylation with low concentrations of diethylpyrocarbonate provided direct evidence of histidyl involvement. The differential response of these proteins to the effects of temperature and urea was, therefore, interpreted based on calculated solvent-accessibilities and surface distributions of His residues, individual His residue pKa values, and specific features of the protein surface structure in the immediate environment of the surface-exposed histidyl residues. Possible interaction mechanisms involved in protein recognition of macromolecular surface-immobilized metal ions are presented.

Two forms of lactoferrin, an intact lactoferrin and a "nicked" but apparently intact (i.e., 78-kDa) form, have been isolated from the urine of preterm infants fed human milk. These two forms of lactoferrin, demonstrated to be entirely of maternal origin, were copurified using affinity columns of immobilized single-stranded DNA-agarose. The relative concentrations of the intact lactoferrin and the "nicked" lactoferrin were determined after denaturation and separation by reverse-phase HPLC. N-terminal sequence analyses showed that the intact 78-kDa form had lost two residues from its N terminus. The nicked 78-kDa form was composed of only two fragments; one fragment was identified as the N terminus of the N-lobe (residues 3-283). The other fragment started with Ser-284 and included the alpha-helical structures at the C terminus of the N-lobe, as well as the entire C-lobe. Although no disulfide bonds connect these two fragments, they were tightly associated in vivo and were not separated in vitro except under denaturing conditions. Limited in vitro digestion of human milk lactoferrin with trypsin produced a nicked, but stable (78-kDa), form of DNA-binding lactoferrin nearly indistinguishable from the isolated urinary lactoferrin, except for the absence of one additional arginine residue at the N terminus of the N-lobe. Residues involved in the stable molecular interaction between fragments were evaluated using data obtained from the high-resolution crystal structure of hololactoferrin. Two features, entirely within the N-lobe, account for the lack of fragment dissociation after cleavage at residue 283 in vivo: an extensive interface at the hinge region behind the iron-binding cleft and an "anchor" sequence traversing the remainder of the N-lobe at 90 degrees relative to the fragment interface. These results document the remarkably limited degradation of absorbed lactoferrin in vivo and suggest that iron-binding activity, receptor-binding properties, and postulated immune cell regulatory functions remain intact.

The origin of intact (78-kD) lactoferrin found in the urine of human milk-fed preterm infants was investigated using human milk containing proteins enriched with [13C]leucine and [15N2]lysine or [2H4]lysine. Mothers of infants selected for the study were infused i.v. with [13C] leucine and [15N2]lysine or [2H4]lysine to label milk proteins. The labeled milk was collected from each mother, pooled, fortified with a lyophilized human milk fraction, and fed to her preterm infant by continuous orogastric infusion for a period of 48 h. Urine was collected from each infant for 96 h. Intact lactoferrin (78 kD) and DNA-binding lactoferrin fragments (51 and 39 kD) were purified from the urine by affinity chromatography on columns of immobilized single-stranded DNA-agarose. The concentration and isotopic enrichment of the intact lactoferrin and DNA-binding fragments were determined separately after their isolation by high-performance reverse-phase (phenyl) chromatography. Mass spectral analyses indicated that the isotopic enrichment of the purified urinary lactoferrin was 87 to 100% of that in the labeled human milk lactoferrin. Similar results were obtained for the isolated DNA-binding lactoferrin fragments. The ratios of isotopically labeled leucine to lysine in the purified milk lactoferrins and urinary lactoferrins were similar for each mother/infant pair. Isotopically labeled lysine, added to the milk as free amino acid, was not incorporated into the purified urinary lactoferrin. These results demonstrate that undegraded (78-kD) lactoferrin of maternal origin is absorbed by the gut and excreted intact in the urine of preterm infants; nearly all of the urinary lactoferrin was of maternal origin. The possible immunoregulatory functions of the absorbed intact, maternal lactoferrin are discussed.

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.

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.

Although some degree of structural homology has been demonstrated among lactoferrins of different species, other reports suggest that these proteins are immunologically distinct. Human, bovine, and porcine lactoferrins were purified to homogeneity from colostral whey by affinity chromatography on immobilized single-stranded DNA. Evidence for shared antigenic determinants among human, bovine, and porcine lactoferrins was demonstrated by Ouchterlony immunodiffusion and immuno "dot" blots using whole antisera and purified Ig directed against each species of lactoferrin. There was no evidence that human transferrin was recognized by any of the lactoferrin-specific antisera evaluated. The degree of cross-reactivity between the lactoferrins and their trypsin digestion products was also evaluated after SDS-PAGE by immunoblotting. These data demonstrate that human, porcine, and bovine lactoferrins share common antigenic determinants and are likely to be more homologous in tertiary structures than suggested previously. Thus, investigations of human or bovine lactoferrin metabolism in infants that are based upon immunologic methods alone should be conducted cautiously in those cases where the presence of both human and bovine lactoferrin is suspected.

The site- or domain-specific immobilization of steroid receptor proteins with preserved structure and function would facilitate the identification and purification of receptor-associated regulatory components and nucleic acids. We have demonstrated previously that restricted surface regions of the estrogen receptor protein contain high affinity binding sites for immobilized Zn(II) ions. Possible conformational changes in receptor at the stationary phase immobilized metal ion interface were evaluated by monitoring alterations in the equilibrium dissociation constant (Kd) for [3H]estradiol. Soluble estrogen receptor proteins (unliganded) present in immature calf uterine cytosol were immobilized via surface-exposed Zn(II)-binding sites to beads of agarose derivatized with iminodiacetate (IDA)-Zn(II) ions. The IDA-Zn(II) bound receptor was incubated with increasing concentrations of [3H]estradiol (0.01-20 nM) in the presence and absence of unlabeled competitor (diethylstilbestrol) to determine the level of specific hormone binding. Steroid-binding experiments were performed in parallel with identical aliquots of soluble receptor. Analyses of the equilibrium binding data revealed the presence of a single class of high-affinity (Kd = 2.44 +/- 1.5 nM, n = 10) steroid-binding sites which were only marginally affected by receptor immobilization via surface-exposed Zn(II) bindings sites (Kd = 2.58 +/- 0.56 nM, n = 4). These data are consistent with the location of surface accessible Zn(II) binding site(s) on the receptor at or near the DNA binding domain which, upon occupancy, do not influence the steroid binding domain.(ABSTRACT TRUNCATED AT 250 WORDS)

In order to evaluate the collective contribution of immunoglobulins to gastrointestinal and immune system development in newborn infants, it would be advantageous to develop a simple and rapid procedure for the selective and quantitative removal of all immunoglobulin classes from colostrum and milk. Toward this end, the major immunoglobulin classes typically present in colostrum (IgM, sIgA, and IgG) have been isolated simultaneously by selective removal from a porcine colostral whey model system using a single-step, preparative scale procedure termed thiophilic adsorption. At neutral pH, the salt-promoted thiophilic affinity of immunoglobulins for the synthetic sulfone-thioether ligands exploits a common (as yet unknown) structural feature of immunoglobulins. The adsorption and recovery procedures operate efficiently under mild buffer conditions permitting the subsequent comparative biochemical analyses of individual immunoglobulin classes for structural and functional heterogeneity. Thus, 1 liter columns of thiophilic adsorbent (T-gel) were employed to obtain pure, structurally intact immunoglobulins from 1 liter batches of porcine colostral whey. The identity and purity of the isolated immunoglobulins were determined under both structure-stabilizing ('native') conditions and denaturing conditions using high-performance size-exclusion chromatography, sucrose density gradient centrifugation, immunodiffusion techniques, SDS-polyacrylamide gradient gel electrophoresis with immunoblotting identification procedures, and two-dimensional electrophoresis. This is the first procedure known to us that allows for the simultaneous one-step isolation (under homologous conditions) of various immunoglobulins with preserved quaternary structure and apparent biological activity.

We have examined the influence of free metal ions on the affinity of structurally-defined proteins and peptides for model surface-immobilized metal ions. The model proteins chosen differed widely in both the type and quantity of surface-accessible electron donor groups. Metal ion affinity chromatography and equilibrium binding analyses demonstrated that the presence of excess free Cu(II) ions did not measurably affect either the affinity or the binding capacity of lysozyme for immobilized iminodiacetate-Cu(II). Similarly, the presence of excess free Cu(II) ions did not detectably affect the chromatographic behavior or measured affinity of either copper-saturated lactoferrin or iron-saturated lactoferrin for the immobilized Cu(II) ions. Its binding capacity however, was diminished. The affinities of small peptides for immobilized Cu(II) ions was found to be related to their number of His residues. Peptides with 0, 1, 2 and 3 His residues were resolved by high-performance immobilized Cu(II) affinity chromatography in both the presence and absence of added Cu(II) ions. In the presence of excess free Cu(II) ions, however, retention (affinities) of these peptides by immobilized Cu(II) ions was increased in relation to their number of His residues. These data demonstrate that protein surface binding sites for free and immobilized metal ions are functionally distinct. The presence of free and/or protein surface-bound metal ions does not preclude interaction with the same immobilized metal ions. Stationary phase immobilized metal ions can be a useful model system through which we can better understand the influence of macromolecular surface-immobilized metal ions on macromolecular recognition events. The significance of these findings are also important to the design of other site-specific and domain-specific affinity reagents involving metal ions.

A new antiestrogen affinity ligand for the covalent labeling of estrogen receptors, [3H]desmethylnafoxidine aziridine, has been used to investigate the salt- and temperature-independent formation of DNA-binding estrogen receptor forms from untransformed (300 kilodaltons) receptor. Calf uterine estrogen receptor proteins labeled with [3H]estradiol or [3H]desmethylnafoxidine aziridine were quantitatively transformed (greater than 90%) to their DNA-binding configuration in low ionic strength buffers by brief exposure to 3 M urea at 0 C. The urea effect was hormone-dependent and partially reversible. The transformed receptors were purified (ca 250-fold) by affinity chromatography on single-stranded DNA-agarose in the continued presence of 3 M urea to prevent transformation reversal. Scatchard analyses revealed a single class of high affinity radioligand binding sites (Kd = 0.34 nM) unchanged by urea-induced transformation and purification. The DNA-binding receptor form labeled with [3H]desmethylnafoxidine aziridine was stable as a probable dimer in 3 M urea with 0.4 M KCl and displayed no evidence of size (Stokes radius 7.3 to 7.5 nm; 4.2 to 4.3 S; Mr = 136,800) heterogeneity. Sodium dodecyl sulfate-polyacrylamide gradient gel electrophoresis indicated the presence of an intact 67 kDa steroid-binding receptor subunit. Reverse-phase chromatography of the covalently labeled receptor on C4 and phenyl stationary phases revealed no evidence of structural heterogeneity. The surface charge of the estrogen- and antiestrogen-receptor complexes, however, was distinctly different in both the presence and absence of 3 M urea. Thus, exposure to urea was an effective salt- and temperature-independent means for achieving the complete transformation of receptor to its stable DNA-binding dimer configuration. The ligand-induced differences in receptor surface charge and the urea effects on DNA-binding (but not hormone-binding) suggest that both electrostatic and hydrophobic or hydrogen bonding receptor domains are influenced by ligand binding.

Physicochemical properties of native and activated (DNA-binding) forms of the glucocorticoid receptor in cytosol prepared from lactating goat mammary tissue have been examined. Under hypotonic conditions the cytosolic receptor sediments at 8.4 S or 9.9 S in the absence or presence of 10 mM molybdate, respectively. The receptor in cytosol, either with or without molybdate elutes from DEAE-cellulose at approximately 200 mM potassium phosphate concentration. Isoelectric focusing reveals that this form of the receptor focuses at pH 5.5. Further, the cytosolic form of the receptor exhibits minimal binding affinity for polyanions such as DNA-cellulose. Its Stokes radius is 77 A and the mol. wt is approximately 331,000. Following exposure to in vitro activating conditions (including elevated ionic strength or temperature), the liganded receptor exhibits much lower affinity for DEAE-cellulose (elution at 35-55 mM potassium phosphate concentration). Other alterations in properties of the activated receptor, after partial purification, include sedimentation at 3.9 S in hypotonic sucrose gradients, binding to polyanions (DNA-cellulose), and an isoelectric point at pH 7.2. This receptor has a Stokes radius of 58 A and a mol wt of 98,000. A degraded form, with a mol. wt of approximately 57,000 and high affinity for polyanions, was the major form of the receptor obtained if appropriate precautions to prevent or remove proteolytic activity were not observed during purification and/or characterization of the activated receptor.