TRACE METALS - FROM DEFICIENCY TO

TOXICITY

TRACE METALS - FROM DEFICIENCY TO

TOXICITYQuest – July 22, 2004

Yeala Shaked, Yan Xu and Francois Morel,

Geosciences Dept, Ecology and Evolutionary Biology Dept. and Princeton Environmental Institute

The transition metal elements V, Mo, W, Mn, Fe, Co, Ni, Cu, and Zn serve as osmotic regulators, as structural glue, and as catalytic centers for hundreds of cellular reactions.

About one third of all characterized enzymes are metalloenzymes.

Trace Metals(red label - metals with known biological role)

TRACE METALS -

FROM DEFICIENCY TO TOXICITY

TRACE METALS -

FROM DEFICIENCY TO TOXICITY

Average elemental composition of a human body

(for a 70 kg adult in units of grams)

O- 45500, C- 12600, H- 7000, N- 2100, Ca- 1050 , P- 700 , S- 175, K- 140 , Cl- 105 , Na- 105, Mg- 35

Fe- 4.2 , Zn- 2.3, Si- 1.4 , F-0.8 , Cu- 0.11 , I -0.03,

Mn -0.02 , Se -0.02, V- 0.02

0

5

10

15

20

25

Low [M] Sub-optimal [M] Optimal [M] High [M]

Growth rate of unicellular algae

Enzyme activity

Tissue function

Deficienc

y

Toxicity

Nutritional supplements

Adaptive strategies- metal replacements, alternative pathways

Chelation therapy

Cellular response- Metal efflux, metal chelation

Metal Toxicity:

1. Non specific binding to biomolecules- destruction of structure and function

2. Replacement of original metal by the toxicant metal, which lack the ability to carry the original function.

Today’s Lecture + lab:

Demonstrating the concept of trace metal deficiency and toxicity

using the Zn metalloenzyme Alkaline Phosphatase

Alkaline Phosphatase –

- Abundance and significance- role of P in the human body and the environment

- Reaction mechanism

- Active center – 2 Zn atoms

- Metal removal by EDTA

- Lab instructions

Alkaline phosphatase (AP)

An enzyme that catalyzes the cleavage of a phosphate group from a variety of compounds.

It is found in most tissues and organs in our body and in most organisms and is important in recycling phosphate within living cells.

It seems to be particularly prevalent in tissues that are transporting nutrients, including intestine and kidney. AP is essential for the deposition of minerals in the bones and teeth among many other functions.

Blood levels tests of AP serve as indicative tool for bones growth, liver cells damage and other diseases.

  2 MG ions    4 ZN ions 2 PO4

E.Coli Alkaline Phosphatase

Role of P in human nutrition

* Humans obtain P from eating plants, and use it to make bones, teeth, and shells. It is also an important constituent of cell membranes, DNA, RNA, and ATP.

*On average, the human body contains 1 3/4 lb of P, with the overwhelming amount found in bones. The remainder is used for other body processes including the metabolism of red blood cells and ATP production. ATP is an energy-rich

compound that fuels activity in the body's cells .

* Phosphorus is excreted as phosphate and organic-P compounds. Soil microbes recycle P for plant uptake, thereby making it available to all animals.

In aquatics environments (mostly lakes) phosphate concentration is often limiting phytoplankton growth.

Organic P compounds are potentially large source of P. However, since microorganisms can not access those compounds, the enzymatic degradation and release of available PO4

3- by Alkaline phosphatase is of large environmental significance.

P in the Environment

Alkaline Phosphatase-

Active site contains two Zn atoms and Mg

Active site of E. coli Alkaline Phosphatase

The Zn atoms play a catalytic role in the reaction

e.g. activating the oxygen of the water or alcohol, and stabilizing the leaving group

The Mg atom has a structural role

p-Nitrophenyl phosphate

+H2O

Alkaline Phosphatase activity assay:

phosphate cleavage from p-Nitrophenyl phosphate

p-Nitrophenolphosphate

+

absorbance at 410nm

AP

The EDTA Molecule

* EDTA or ethylenediaminetetraacetic acid is a novel molecule for complexing metal ions.  * It is a polyprotic acid containing four carboxylic acid groups (acidic hydrogens are red) and two amine groups with lone pair electrons (green dots). 

* On a worldwide basis over 100,000 metric tons are produced annually. Because of its strong complexing ability for most metal ions, it is used in the food industry as a sequestering agent.  The complexing of the metal ion may prevent further reactions, such as binding metals that are cofactors for enzymes, or just remove a metallic taste, such as metal contamination added during processing.  

Lab plan:

Goal- Demonstrating the concept of metal deficiency and toxicity using E. coli Alkaline phosphatase.

Enzyme activity assay (color forming reaction) with different levels of Zn.

Name MW Concentration in stock

weight Volume (ml)

Note

Tris-base 121.14 0.1 M 6.057g 500ml Adjust pH with HCl to 8.0

Ecoli alkaline phosphatase

80,000 11.8ul 200ml In Tris buffer

4-Nitrophenyl phosphate disodium salt hexahydrate (pNPP)

371.14 3 mM 0.2227g 200ml In Tris buffer

Ethylenediaminetetraacetic acid tetrasodium salt hydrate (EDTA)

452.23 60 mM 5.427g 200ml In H2O

Zinc chloride 136.3 0.3 M 0.4089g 10ml In HCl,

prepare later Cadmium chloride 228.4 0.3 M 0.6852g 10ml In HCl,

prepare later

pH buffer

enzyme

substrate

Metal chelator

Metals

36 ul

Lab instructions:

Step # Cuvette

number 1 2 3 4 5 6 7 8 9

Enzyme 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 EDTA 1.0 1.0 1.0 1.0 1.0 H2O 1.0 1.0 1 Mix the solution and wait for 45 min.

Prepare ZnSO4 and CdCl2 solution in the meantime ZnSO4 1.0 CdCl2 1.0 2 Mix the solution,

and then add pNPP, 1 ml each and mix them again

PNPP 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 1.0 Observe the color change for 10 min and record the rate of

yellow color formation using color code (0-5)* Result 1

Color code (0-5)

Refer to questions 1-6

3 ZnSO4 0.1 0.2 CdCl2 0.2 1.0 Mix the solution

and observe the color change for 10 min

Result 2 Color code (0-5)

Refer to questions 7-10