Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol

Doyle, Colleen; Naser, Dalia; Bauman, Heather A.; Rumfeldt, Jessica A.O.; Meiering, Elizabeth M.

doi:10.1016/j.ab.2019.03.007

Cited by 14 publications

(7 citation statements)

References 39 publications

(91 reference statements)

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“…In the control titration, adding Cu(II) to apo-PAR decreased the apo-PAR absorbance at 400 nm and concomitantly increased the Cu(II)-PAR absorbance at 512 nm (Figure 10A). As anticipated [58], this titration produced an end point at 1:1 Cu(II):PAR (Figure S9A). Adding Cu(II) into apo-PAR in the presence of 20 molar equiv.…”

Section: Molecular Basis For the Observed Action Of Hst5 As A Strong Cu Buffersupporting

confidence: 72%

“…Since both Hst5 and NTA have weak optical signals (Figure S8), this log K Cu value was re-evaluated here using PAR as an intensely coloured mediator 50 between NTA and Hst5. The control titration confirmed that adding Cu(II) decreased the solution absorbance of apo- PAR at 400 nm and concomitantly increased that of the Cu(II)-PAR complex 51 at 512 nm (Figure 10A, Figure S9A). PAR was then competed with 20 molar equiv.…”

Section: Resultsmentioning

confidence: 67%

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The role of metal binding in the function of the human salivary antimicrobial peptide histatin-5

Stewart

Hong

Holmes

et al. 2022

Preprint

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The family of human salivary histidine-rich peptides known as histatins bind zinc (Zn) and copper (Cu), but whether they contribute to nutritional immunity by influencing Zn and/or Cu availability has not been examined. We hypothesised that histatin-5 (Hst5) limits Zn availability (and promotes bacterial Zn starvation) and/or raises Cu availability (and promotes bacterial Cu poisoning). To test this hypothesis, Group A Streptococcus (GAS), which colonises the human oropharynx, was used as a model bacterium. Contrary to our hypothesis, Hst5 did not strongly influence Zn availability. This peptide did not induce expression of Zn uptake genes in GAS, nor did it suppress growth of an ΔadcAI mutant strain that is impaired in Zn uptake. Equilibrium competition measurements confirmed that Hst5 binds Zn weakly and does not compete with the high-affinity Zn uptake protein AdcAI for binding Zn. By contrast, Hst5 bound Cu with a high affinity and strongly influenced Cu availability. However, contrary to our hypothesis, Hst5 did not promote Cu toxicity. Instead, this peptide suppressed expression of Cu-inducible genes in GAS, stopped intracellular accumulation of Cu, and rescued growth of a ΔcopA mutant strain that is impaired in Cu efflux in the presence of added Cu. These findings led us to propose a new role for Hst5 and salivary histatins as major Cu buffers in saliva that reduce the potential negative effects of Cu exposure to microbes. We speculate that histatins promote oral and oropharyngeal health by contributing to microbial homeostasis in these host niches.

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Section: Molecular Basis For the Observed Action Of Hst5 As A Strong Cu Buffersupporting

confidence: 72%

Section: Resultsmentioning

confidence: 67%

The role of metal binding in the function of the human salivary antimicrobial peptide histatin-5

Stewart

Hong

Holmes

et al. 2022

Preprint

View full text Add to dashboard Cite

show abstract

“…Each of the heat-treated NCp7 samples were dialyzed overnight against 1×HEPES and diluted to 25 µM. The Zn 2+ content of each protein was analyzed via a spectrophotometric method using the 4-(2-pyridylazo) resorcinol (PAR) assay according to the protocol described by Doyle et al [26]. The Zn 2+ content of 0 NCp7, NCp7 and 2 NCp7 were also confirmed using inductively coupled plasma-optical emission spectrometer (ICP-OES) on the Spectro analytical instruments (Kleve, Germany).…”

Section: Determination Of Zn 2+ Contentmentioning

confidence: 99%

The Zinc Content of HIV-1 NCp7 Affects Its Selectivity for Packaging Signal and Affinity for Stem-Loop 3

Wang

Guo

Wang

et al. 2021

Viruses

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The nucleocapsid (NC) protein of human immunodeficiency (HIV) is a small, highly basic protein containing two CCHC zinc-finger motifs, which is cleaved from the NC domain of the Gag polyprotein during virus maturation. We previously reported that recombinant HIV-1 Gag and NCp7 overexpressed in an E. coli host contains two and one zinc ions, respectively, and Gag exhibited much higher selectivity for packaging signal (Psi) and affinity for the stem-loop (SL)-3 of Psi than NCp7. In this study, we prepared NCp7 containing 0 (0NCp7), 1 (NCp7) or 2 (2NCp7) zinc ions, and compared their secondary structure, Psi-selectivity and SL3-affinity. Along with the decrease of the zinc content, less ordered conformations were detected. Compared to NCp7, 2NCp7 exhibited a much higher Psi-selectivity and SL3-affinity, similar to Gag, whereas 0NCp7 exhibited a lower Psi-selectivity and SL3-affinity, similar to the H23&H44K double mutant of NCp7, indicating that the different RNA-binding property of Gag NC domain and the mature NCp7 may be resulted, at least partially, from their different zinc content. This study will be helpful to elucidate the critical roles that zinc played in the viral life cycle, and benefit further investigations of the functional switch from the NC domain of Gag to the mature NCp7.

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“…Due to its accessibility, sensitivity, and suitability, flame atomic absorption spectrophotometry was utilized in the current study. The Zinc metal bonded with many enzyme systems such as metalloenzyme and enzyme activators [6][7][8]. Zinc was determined in human blood for medical diagnosis [9], while in the environmental samples (water and soil), the Zinc concentration was determined to access the pollution index [10].…”

Section: Introductionmentioning

confidence: 99%

Determination of the Zinc level in Environmental and Biological Samples in Baiji City by Atomic Absorption Spectrophotometer

Hasan¹

2022

Tikrit J. Pure Sci.

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This study was conducted to measure Zinc levels in environmental and biological samples. The method was tested with three types of samples, including soil, water, and human blood serum. In this method, 50 samples from each soil, water, and human blood serum were used. The water and soil samples were collected from the Baiji oil refinery, while the human blood serum was collected from the patients in the Baiji hospital. The total Zinc concentration in the environmental and biological samples was measured by atomic absorption spectrophotometry (AAS). The results of the Zinc determination were obtained from the samples taken from the soil for each site under examination. The following values showed the mean average concentration for each site, respectively. site 1 (15±3.5), site 2 (11.5±1.1), site 3 (14.5) ±0.5), and site 4 (13.8±3.8) µg/g. Moreover, the Zinc concentration in the water samples was between 3-14.3 µg/ mL. The Zinc concentration in the blood serum samples of the control and the healthy group was 598 ± 32 µg/mL, and in the patient group was 450 ± 16 µg/mL.

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Spectrophotometric method for simultaneous measurement of zinc and copper in metalloproteins using 4-(2-pyridylazo)resorcinol

Cited by 14 publications

References 39 publications

The role of metal binding in the function of the human salivary antimicrobial peptide histatin-5

The role of metal binding in the function of the human salivary antimicrobial peptide histatin-5

The Zinc Content of HIV-1 NCp7 Affects Its Selectivity for Packaging Signal and Affinity for Stem-Loop 3

Determination of the Zinc level in Environmental and Biological Samples in Baiji City by Atomic Absorption Spectrophotometer

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