Lead-binding Proteins Research Articles

Rapid determination of toxic and essential metal binding proteins in biological samples by size exclusion chromatography-inductively coupled plasma tandem mass spectrometry

Metalloproteins binding with trace elements play a crucial role in biological processes and on the contrary, those binding with exogenous heavy metals have adverse effects. However, the methods for rapid, high sensitivity and simultaneous analysis of these metalloproteins are still lacking. In this study, a fast method for simultaneously determination of both essential and toxic metal-containing proteins was developed by coupling size exclusion chromatography (SEC) with inductively coupled plasma tandem mass spectrometry (ICP-MS/MS). After optimization of the separation and detection conditions, seven metalloproteins with different molecular weight (from 16.0 to 443.0 kDa) were successfully separated within 10 min and the proteins containing iron (Fe), copper (Cu), zinc (Zn), iodine (I) and lead (Pb) elements could be simultaneously detected with the use of oxygen as the collision gas in ICP-MS/MS. Accordingly, the linear relationship between log molecular weight and retention time was established to estimate the molecular weight of unknown proteins. Thus, the trace metal and toxic metal containing proteins could be detected in a single run with high sensitivity (detection limits in the range of 0.0020–2.5 μg/mL) and good repeatability (relative standard deviations lower than 4.5 %). This method was then successfully used to analyze metal (e.g., Pb, Zn, Cu and Fe) binding proteins in the blood of Pb-intoxicated patients, and the results showed a negative correlation between the contents of zinc and lead binding proteins, which was identified to contain hemoglobin subunit. In summary, this work provided a rapid and sensitive tool for screening metal containing proteins in large number of biological samples.

Identification of lead-binding proteins as carriers and potential molecular targets associated with systolic blood pressure

Lead (Pb) exposure is well recognized as a significant environmental factor associated with the high incidence of cardiovascular diseases. However, the carriers and molecular targets of Pb in human blood remain to be understood, especially for a real Pb exposure scenario. In this study, a total of 350 blood samples were collected from the smelting workers and systematically analyzed using metallomics and metalloproteomics approaches. The results showed that the majority of Pb (∼99.4%) could be presented in the blood cells. Pb in the cytoplasm of blood cells accounted for approximately 83.1% of the total blood Pb, with nearly half of Pb being bound to proteins. Pb-binding proteins in the blood of workers were identified as hemoglobin, catalase, haptoglobin, δ-aminolevulinic acid dehydratase, and peroxiredoxin-2. Multiple linear regression analysis demonstrated that higher levels of Pb bound to proteins (Mix-bound Pb and Protein-bound Pb) were positively associated with higher systolic blood pressure (p < 0.05). However, the association between blood lead level, Pb levels in the blood cells and systolic blood pressure was not observed (p > 0.05). This study suggested that Pb bound to proteins could be a suitable biomarker for indicating the potential risk of occupational hypertension.

Sensitive determination of metalloprotein in salt-rich matrices by size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry

Metalloproteins play crucial and distinct roles in a variety of biological processes that rely heavily on the metal ions and various proteins. However, there is still a lack of method for rapid analysis of metalloproteins in complex samples, especially in salt-rich matrices. In this study, a sensitive method for separation and determination of metalloproteins in salt-rich matrices was developed based on the size exclusion chromatography coupled with inductively coupled plasma-mass spectrometry (SEC-ICP-MS), combining with the high matrix introduction (HMI) mode, which is quite essential for biological system. The separation conditions of the SEC-ICP-MS system were optimized by using four iodine labeled proteins with different molecular weights, including bovine serum albumin (BSA, 66.0 kDa), ovalbumin (OVA, 44.0 kDa), carbonic anhydrase (CA, 29.0 kDa) and ribonuclease A (RA, 13.7 kDa). After optimization, four iodine labeled proteins and iodine ions were successfully separated within 30 min by using 10 mmol/L HEPES and 40 mmol/L Na2SO4 (pH=7.0) as mobile phase and a linear relationship between log molecular weight and retention time was established. The relative standard deviations (RSDs, n = 5) of the retention time and peak areas for the four iodine labeled proteins were in the range of 0.2–0.9% and 3.3–7.7%, respectively, suggesting good precision and repeatability. Then the proposed method was successfully applied to the rapid separation and detection of lead-binding proteins in real biological tissue samples.

Recombinant expression and purification of a functional bacterial metallo-chaperone PbrD-fusion construct as a potential biosorbent for Pb(II)

PbrD is a lead (II) binding protein encoded by the pbr lead resistance operon found exclusively in Cupriavidus metallidurans CH34. Its ability to sequester Pb(II) shows potential for it to be developed as a biosorbent for Pb in the bioremediation of contaminated wastewaters. In this study the pbrD gene from C. metallidurans CH34 was transformed and overexpressed in Escherichia coli BL21 (DE3) using the pET32 Xa/Lic vector. Optimal expression of recombinant (r)PbrD (∼50 kDa) was achieved post-induction with IPTG within inclusion bodies (IBs). Inclusion bodies were solubilised by denaturation and purified by Ni-NTA affinity chromatography. The purified denatured protein containing the N-terminal Trx•Tag™, His•Tag® and S®Tag™ was refolded in vitro via dialysis to a biologically functional form. Circular dichroism spectra of refolded rPbrD-fusion protein indicated a high degree of turns, β-sheets and 310 helices content and tryptophan fluorescence showed a structural conformational change in the presence of Pb(II). Refolded rPbrD-fusion protein bound 99.7% of Pb(II) when mixed with lead nitrate in ten-fold increasing concentrations. Adsorption isotherms including Langmuir, Freundlich, Temkin and Dubinin-Radushkevich models were applied to determine the biosorption mechanism. A biologically functional rPbrD-fusion protein has potential application in the development of a biosorbent for remediation of Pb(II) from wastewater.

Using Recombinant E. coli Displaying Surface Heavy Metal Binding Proteins for Removal of Pb2+ from Contaminated Water

Water pollution remains a serious problem with economic and public health concerns worldwide. Lead (Pb2+) is one of the dangerous metals related to chronic diseases and is responsible for many deaths around the world. Despite the advances in technologies for removal of heavy metals e.g., Pb2+ from water, all current techniques have shown some limitations that obstructed their application. Bearing in mind that there is a need to develop a novel technique for removal of heavy metals from water, we developed a quick, specific and efficient method for removal of Pb2+ from water using dead cells of recombinant Escherichia coli. Recombinant E. coli were engineered to display metallothionein (SmtB) and lead binding protein (PbrR) onto outer membrane. DNA fragments encoding these proteins were fused to DNA fragment encoding β- domain of antigen 43 (Ag43) for translocation of both heavy metal binding proteins. The resultant recombinant E. coli exhibited a capability to adsorb Pb2+ successfully from water samples containing 100 mg/L of Pb2+, and concentrations of Pb2+ reached to undetectable level after 18 hours. Heat-inactivated E. coli displaying PbrR and SmtB on outer membrane showed comparable removal efficiencies to live E. coli cells. These observations suggest that our method can be used as a promising, specific and efficient approach for removal of Pb2+ from contaminated water.

Irregular gout

Irregular gout

Lead-Binding Proteins: A Review

Lead-binding proteins are a series of low molecular weight proteins, analogous to metallothionein, which segregate lead in a nontoxic form in several organs (kidney, brain, lung, liver, erythrocyte). Whether the lead-binding proteins in every organ are identical or different remains to be determined. In the erythrocyte, delta-aminolevulinic acid dehydratase (ALAD) isoforms have commanded the greatest attention as proteins and enzymes that are both inhibitable and inducible by lead. ALAD-2, although it binds lead to a greater degree than ALAD-1, appears to bind lead in a less toxic form. What may be of greater significance is that a low molecular weight lead-binding protein, approximately 10 kDa, appears in the erythrocyte once blood lead exceeds 39 μg/dL and eventually surpasses the lead-binding capacity of ALAD. In brain and kidney of environmentally exposed humans and animals, a cytoplasmic lead-binding protein has been identified as thymosin β4, a 5 kDa protein. In kidney, but not brain, another lead-binding protein has been identified as acyl-CoA binding protein, a 9 kDa protein. Each of these proteins, when coincubated with liver ALAD and titrated with lead, diminishes the inhibition of ALAD by lead, verifying their ability to segregate lead in a nontoxic form.

A lead-absorbing protein with superoxide dismutase activity from Streptomyces subrutilus

A lead binding protein was purified from the culture filtrate of Streptomyces subrutilus P5. The subunit and native molecular weights were estimated to be 28 and 55 kDa, respectively, indicating that the protein was composed of two identical subunits. The inhibition pattern, the metal content analysis and the EPR spectrum confirmed that the protein was a superoxide dismutase containing Fe and Zn (FeZnSOD). The protein precipitated immediately upon mixing with lead ions and the saturation number of lead ions was about 1100 lead atoms per subunit. Using this property, lead ions could be effectively removed from solutions.

A lead-absorbing protein with superoxide dismutase activity from Streptomyces subrutilus.

A lead binding protein was purified from the culture filtrate of Streptomyces subrutilus P5. The subunit and native molecular weights were estimated to be 28 and 55 kDa, respectively, indicating that the protein was composed of two identical subunits. The inhibition pattern, the metal content analysis and the EPR spectrum confirmed that the protein was a superoxide dismutase containing Fe and Zn (FeZnSOD). The protein precipitated immediately upon mixing with lead ions and the saturation number of lead ions was about 1100 lead atoms per subunit. Using this property, lead ions could be effectively removed from solutions.

Detection of novel ALAD gene polymorphisms using denaturing high-performance liquid chromatography.

Denaturing high-performance liquid chromatography (DH-PLC), which is based on the separation of mismatched DNA heteroduplexes, is one of the most promising techniques for detecting nucleotide polymorphisms. Lead is an important environmental toxicant that can impair the cardiovascular, central nervous, renal, reproductive, and hematologic systems. Here we compare the sensitivity and efficiency of DNA polymorphism detection in the delta-aminolevulinate dehydratase (ALAD) gene encoding the principal lead-binding protein in humans by means of DHPLC and direct DNA sequencing of polymerase chain reaction amplicons. In a sample of 48 unrelated Chinese women, five novel mutations were discovered in intron 6 (G13298C). exon 7 (C13348T), intron 8 (C13847T), intron 12 (C15096T), and the 3' untranslated region of exon 13 (A15762C). The allele frequencies of C13298, T13348, T13847, T15096, and C15762 alleles were 21.3%, 2.3%. 82.1%, 62.5%, and 1.1%, respectively. All five mutations were detected by both DHPLC and direct DNA sequencing. No previously reported missense ALAD mutations were found in this Chinese population. Our study confirms that DHPLC provides an accurate method for the rapid identification of single nucleotide polymorphisms.

Roles of lead-binding proteins in mediating lead bioavailability.

The intracellular bioavailability of lead (Pb) at low dosage levels in major target organs such as the kidney and brain appears to be largely determined by complexation with a group of low molecular weight proteins. These proteins are rich in aspartic and glutamic dicarboxyl amino acids. The proteins are chemically similar but not identical across all species examined to date and the brain protein appears to be different from that found in the kidney. These proteins possess dissociation constant values for Pb on the order of 10(-8) M and appear to normally bind zinc. In rats, these proteins attenuate the Pb inhibition of the heme pathway enzyme delta-aminolevulinic acid dehydratase by a mechanism involving both Pb chelation and zinc donation to this highly Pb-sensitive zinc-dependent enzyme. Other studies in rats have shown that the kidney protein facilitates the intranuclear movement of Pb in vitro followed by chromatin binding, suggesting that this protein may be involved in alterations of the pathognomonic Pb intranuclear inclusion bodies in renal gene expression associated with the mitogenic effects of Pb in the kidney.

High-affinity renal lead-binding proteins in environmentally-exposed humans

Chronic low level lead (Pb) exposure is associated with decrements in renal function in humans, but the molecular mechanisms underlying toxicity are not understood. We investigated cytosolic Pb-binding proteins (PbBP) in kidney of environmentally-exposed humans to identify molecular targets of Pb and elucidate mechanisms of toxicity. This study is unique in that it localized PbBPs based on physiologic Pb that was bound in vivo. Two Pb-binding polypeptides were identified, thymosin β 4 (T β 4, 5 kDa) and acyl-CoA binding protein (ACBP, 9 kDa, also known as diazepam binding inhibitor, DBI). These polypeptides, which have not been previously recognized for their metal-binding capabilities, were shown to bind Pb with high affinity ( K d≈14 nM) and to account for an estimated >35% of the total Pb in kidney cortex tissue. Both T β 4 and ACBP (DBI) occur across animal species from invertebrates to mammals and in all major tissues, serving multiple possible functions (e.g. regulation of actin polymerization, calmodulin-dependent enzyme activity, acyl-CoA metabolism, GABA-A/benzodiazepine receptor modulation, steroidogenesis, etc.). Thus, these data provide the first evidence of specific molecular targets of Pb in kidney of environmentally-exposed humans, and they suggest that low-level Pb toxicity may occur via alteration of T β 4 and ACBP (DBI) function in renal and other tissues, including the central nervous system.

Lead-binding proteins - a way to understand lead toxicity?

espite the vast liter ature on lead toxicity [1], little is known about the biochemical mechanisms responsible for the toxicity of lead and other metals. The general assumption is that lead interacts with the function of enzymes, signal systems and membranes, probably by binding to certain sites of proteins. Recent studies in the labor atory in Lund have shown a remarka ble specificity for the binding of lead to certain red blood cell proteins, such as δaminolevulinic acid dehydratase (ALAD) [2]. This binding causes inhibition of the enzymatic activity. The inhibition of ALAD activity by lead has been known since the 1960s, but there are also other, as yet unidentified, lead-binding proteins. By identifying the proteins prone to bind lead , we may be able to understand lead toxicity better; identify enzymatic or other biological processes affected; find markers of lead exposure and effects; identify individuals especially sensiti ve to the metal; figure out candidates for antidotes; and under stand how lead is transported within the cell and in the bod y. The same principle should hold true for other toxic metals as well.

Lead-binding proteins - a way to understand lead toxicity?

Lead-binding proteins - a way to understand lead toxicity?

The chemical speciation of aluminium and silicon in human serum A. Sanz-Medel M 81 Lead-binding proteins - a way to understand lead toxicity?

The chemical speciation of aluminium and silicon in human serum A. Sanz-Medel M 81 Lead-binding proteins - a way to understand lead toxicity?

The chemical speciation of aluminium and silicon in human serum A. Sanz-Medel M 81 Lead-binding proteins - a way to understand lead toxicity?

The chemical speciation of aluminium and silicon in human serum A. Sanz-Medel M 81 Lead-binding proteins - a way to understand lead toxicity?

Studies on lead-binding protein and interaction between lead and selenium in the human erythrocytes.

To clarify the function of Pb-binding protein and the interaction between lead and selenium in human erythrocytes, the following experiments were performed. (a) blood from a man who has not been exposed to lead occupationally, (b) blood from lead-exposed workers and (c) blood from control subjects working in the same plant were used. (a) was incubated with Pb and/or Se. Hemolysates of rinsed erythrocytes were applied onto a Bio-gel A 1.5 m column and eluted by 40 mM Tris-HCl buffer (pH 7.0). As the results, (1) Pb in erythrocytes was found in ALAD fraction. When Pb was added in vitro at 1.25 microM of final concentration, Pb content of ALAD fraction increased and the other Pb-containing fraction appeared at the position of a smaller molecular size than that of Hb fraction. When Pb dose added was increased to 2.5 or 10 microM, Pb contents in the newly appeared fraction was larger than that in ALAD fraction. The affinity of Pb with the protein in the new fraction was weak, and Pb was released from the protein by ultrafiltration. (2) There were main and sub-peaks containing Pb in erythrocytes from Pb-workers; the former was ALAD fraction and the others were smaller molecules than ALAD. The peak of Pb observed with the blood from a worker who has worked at the same plant for more than 20 years (chronic exposure) corresponded with the new peak which appeared in vitro Pb exposure. (3) As to the interaction between Pb and Se, there was no effect of Se added in vitro on the position of Pb-containing fraction and on Pb amounts in the chromatographic profile. When Se was incubated with blood, however, coexistence of Pb made Se concentration in erythrocytes high compared with the case of Se alone.

Lead binding to delta-aminolevulinic acid dehydratase (ALAD) in human erythrocytes.

Over 99% of the lead present in blood is usually found in erythrocytes. To investigate the nature of this selective accumulation of lead in erythrocytes, the specific binding of lead to proteins in human erythrocytes was studied using liquid chromatography coupled to inductively coupled plasma mass spectrometry (LC-ICP-MS). The principal lead-binding protein had a mass of approximately 240 kDa, and adsorption to specific antibodies showed that protein was delta-aminolevulinic acid dehydratase (ALAD). Thus, the previous notion that lead in erythrocytes was bound primarily to haemoglobin has to be revised. Furthermore, in lead-exposed workers, the percentage of lead bound to ALAD was influenced by a common polymorphism in the ALAD gene. Specifically, in seven carriers of the ALAD2 allele, 84% of the protein-bound lead recovered was bound to ALAD compared to 81% in seven homozygotes for the ALAD1 allele whose erythrocytes were matched for blood-lead concentration. The small difference was statistically significant in Wilcoxon matched-pairs signed-rank test (P = 0.03). No ALAD allele-specific difference in ALAD-bound lead was found among 20 unexposed controls. Perhaps the difference in ALAD-bound lead can provide an explanation for the previously reported finding of higher blood-lead levels among carriers of the ALAD2 allele than among ALAD1 homozygotes in lead-exposed populations.

Iron Deficiency Anemia Alters Lead Distribution in Rats † 682

Background: Lead is bound mainly to intracellular protein. Iron deficiency anemia (IDA) may alter red cell protein content and affect blood lead levels. Objective: To determine the effect of IDA on whole blood lead (WBL) levels for an equivalent body burden of lead. Methods: 12 weanling rats were divided into 2 groups receiving either a normal diet (50 PPM iron) or a low iron diet (<5 PPM) otherwise equivalent. After 4 weeks, rats were weighed, jugular veins accessed, and baseline spun hematocrit (Hct) and WBL measured by stable isotope dilution-inductively coupled plasma mass spectrometry. Lead acetate (5 mg/kg body weight) was administered intravenously, and rats continued on their respective diets for 7 days to allow for lead tissue distribution, then sacrificed for tissue lead measurement. Results: Hct's were different: iron replete 47± 5% SD; iron deficient 29± 3% (p= 0.0001). Baseline WBL's were not different: iron replete 0.23 ± 0.18μg/dl SD; iron deficient 0.11 ± 0.07 μg/dl (p=0.2). WBL and tissue lead levels 7 days after lead injection are provided below.Table Conclusion: Although tissue levels were not different, IDA altered lead distribution such that increased lead was found in blood. Blood levels in IDA may represent different tissue burdens than in iron repletion. Possible explanations include increased RBC lead binding protein or changes in RBC membrane transport of lead.

Lead-binding proteins in brain tissue of environmentally lead-exposed humans

This study reports the partial purification and characterization of cytosolic lead binding proteins (PbBPs) in human brain tissue of environmentally Pb-exposed subjects. The isolated proteins were initially characterized based upon the presence of endogenously associated Pb. Following partial purification (Sephadex G-75 and A-25 DEAE anion-exchange chromatography), the isolated PbBPs (contained within a single DEAE peak) showed a single class of high affinity binding sites with an apparent K d of 10 −9 M, based upon competition assays using radioactive 203Pb and Hill and Scatchard analysis. The presence of endogenously bound Pb with the isolated proteins indicated the association of Pb with the protein(s) in vivo in these environmentally Pb-exposed subjects, since the samples were prepared in an ultraclean lead analysis laboratory. Moreover, the persistence of Pb-protein binding throughout the initial two steps (Sephadex G-75 and A-25 DEAE) of the purification scheme is consistent with the high affinity and stability of binding measured with the radiolead competition assays. The DEAE isolated PbBPs were further purified by denaturing reversed-phase HPLC analysis, resulting in the isolation of two proteins, thymosin β 4 (5 kDa, p I 5.1) and a second as yet unidentified protein with an approximate molecular mass of 20 kDa and a pI of 5.9. Qualitative 203Pb-binding analysis of these HPLC purified proteins suggested that they may be primarily responsible for the observed Pb binding in the single DEAE peak. Nearly identical results were obtained in brain cytosols from male and female, and young and adult individuals, although further quantitative analyses are needed to investigate possible sex and age relationships. These data are significant because they contribute to a better understanding of the presence of PbBPs in a sensitive target organ for Pb toxicity in humans, suggesting a possible role of these or similar proteins as sensitive biomarkers of Pb exposure and toxicity.