Transcript Shakedpresentation

TRACE METALS FROM DEFICIENCY TO TOXICITY
Quest – July 22, 2004
Yeala Shaked, Yan Xu and Francois Morel,
Geosciences Dept, Ecology and Evolutionary Biology Dept. and
Princeton Environmental Institute
Trace Metals
(red label - metals with known
biological role)
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 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
25
Growth rate of 20
unicellular algae
Nutritional
supplements
Chelation therapy
Cellular responseMetal efflux, metal
chelation
Adaptive strategiesmetal replacements,
alternative pathways
15
Enzyme activity
10
Tissue function
5
0
Low [M]
Sub-optimal [M]
Optimal [M]
High [M]
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
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.
P in the Environment
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 PO43by Alkaline phosphatase is of large environmental significance.
Alkaline PhosphataseActive 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
Alkaline Phosphatase activity assay:
phosphate cleavage from p-Nitrophenyl phosphate
p-Nitrophenyl phosphate
AP
+
phosphate
H 2O
p-Nitrophenol
+
absorbance at 410nm
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
Tris-base
121.14
enzyme
Ecoli alkaline
phosphatase
80,000
substrate
4-Nitrophenyl phosphate
disodium salt hexahydrate
(pNPP)
Ethylenediaminetetraacetic
acid tetrasodium salt
hydrate (EDTA)
371.14
pH buffer
Metal
chelator
Metals
Zinc chloride
Cadmium chloride
Concentration weight
in stock
0.1 M
6.057g
Volume
(ml)
500ml
Note
Adjust pH
with HCl to
8.0
In Tris
buffer
In Tris
buffer
36 ul
11.8ul
200ml
3 mM
0.2227g
200ml
452.23
60 mM
5.427g
200ml
In H2O
136.3
0.3 M
0.4089g
10ml
228.4
0.3 M
0.6852g
10ml
In HCl,
prepare later
In HCl,
prepare later
Lab instructions:
Step # Cuvette
number
Enzyme
EDTA
H2 O
1
1
2
3
4
5
6
7
8
9
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
Mix the solution and wait for 45 min.
Prepare ZnSO4 and CdCl2 solution in the meantime
1.0
ZnSO4
CdCl2
2
PNPP
1.0
Mix the solution,
and then add pNPP, 1 ml each and mix them again
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
CdCl2
0.1
0.2
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