Determination of Xeno-Estrogens Using a Modified ...

Determining the binding constants of xeno-estrogens using fluorescence methods Outline Introduction Current Determination Methods Estrogen Receptor & Xenobiotic Estrogens Chemical & Biological Fluorescence Method Protein Production & Purification Detection Analysis

Conclusions What is Estrogen? Female Hormone Regulation of the Menstrual Cycle and related female childbearing organs (such as the uterus and ovaries) Maintain healthy bones and heart Required for development of the breast OH HO Hormone Action Figure 1. The mechanism by which estrogen acts to regulate gene expression.

The Human Estrogen Receptor Belongs to the nuclear receptor superfamily Regulates hormone action Can be divided into three separate functional domains Transactivation DNA Binding Ligand Binding Ligand Binding Domain Figure 2. Three dimensional representation of the ligand binding domain of the human estrogen receptor. Xenobiotic Estrogens Environmental estrogens

What are they? Naturally occurring or synthetic chemicals that can act like human estrogen made by the ovary Mimic the effect of estrogen What are some examples? Several Pesticides(including DDT) Food Preservatives (BHT and BHA) Industrial Detergent by-products (nonylphenol) Red Dye #3 Xenobiotic Estrogens Environmental Estrogens

What are they? Naturally occurring or synthetic chemicals that can act like human estrogen made by the ovary Mimic the effect of estrogen What are some examples? Dioxins Several Pesticides(including DDT) Food Preservatives (BHT and BHA) Industrial Detergent by-products (nonylphenol) Diethylstilbestrol (DES) From structural classes such as: Pyrazole Stilbestrol

Isoflavones benzofurans Xenobiotic Estrogens What is their link to altered fertility and breast cancer? Some xenoestrogens increase cell division and thus may contribute to breast cancer Cl OH Cl C Cl Cl HO Cl Nonylphenol

BHT o,p-DDT Outline Introduction Current Determination Methods Estrogen Receptor & Xenobiotic Estrogens Chemical & Biological Fluorescence Method Protein Production & Purification Detection

Analysis Conclusions Current Determination Methods Chemical EPAs MRM (multiple residue method) GC-MS Method accurate Difficult to identify Novel Estrogen Mimics

Biological MCF7 Cells Sensitive to estrogen Growth occurs at increased rates in the presence of estrogen mimics Time consuming Outline Introduction Current Determination Methods

Estrogen Receptor & Xenobiotic Estrogens Chemical & Biological Fluorescence Method - Protein Production & Purification Detection Analysis Questions Introduction to Proteins Proteins play a vital role in all living systems Biological function of a protein is determined by its three dimensional

structure Ability to interact with molecules is dependent on structure and conformational flexibility O OH NH2 O NH2 HO HO NH2 alanine The 20 Amino Acids

OH O NH2 leucine O valine O H2N isoleucine O glycine H O HO OH N H

tryptophan NH2 NH2 O O OH H2N OH phenylalanine tyrosine O N HN H2N

OH NH2 histidine H2N O H2N O - O O OH OH serine OH

arginine O O- HO NH2 O aspartate H2N OH NH2 OH O NH OH

lysine NH2 H N glutamate O NH2 O NH2 NH2 HO HO O

O OH cysteine S HO H2N NH2 methionine SH H N proline O OH OH O

NH2 O asparagine O threonine H2N glutamine O Fundamentals of protein structure Some Residues buried other are not Aromatic Amino Acids O OH NH2 N H

tryptophan HO NH2 O O OH tyrosine H2N OH phenylalanine Production of Hormone Binding Domain of the Estrogen Receptor Start with Bacterial Plate transformed with pMAL-HBD Inoculate Large Liquid Culture and grow to OD600 = 0.8

Induce via IPTG and Grow Overnight @ 25oC Grow Overnight @ 37oC Inoculate Liquid culture with a single colony Purification of Hormone Binding Domain of the Estrogen Receptor Purify ER-MBP from bacterial proteins using affinity column chromatography Use Lysozyme to rupture cells and release proteins Spin to separate bacterial debris from HBD-MBP Collect Fractions

& identify purified product Purification of Hormone Binding Domain of the Estrogen Receptor Hydroxylamine Fraction containing purified fusion protein Separate the HBD from MBP via column chromatography (either size exclusion or ion-exchange) MBP HBD The purified fusion protein is cleaved with hydroxylamine

Analyze and save purified HBD Results of SDS-Page Purified Protein Outline Introduction Current Determination Methods Estrogen Receptor & Xenobiotic Estrogens Chemical & Biological

Fluorescence Method - Protein Production & Purification Detection Analysis Questions Shine Shine UV UV light light Fluoresce Blue light Large Conformational change takes place Conformational change 550 500

Without Estrogen Flourescence Intensity 450 400 350 300 250 With Estrogen 200 150 100 300 310 320 330 340

350 Wavelength 360 370 380 Can get binding data! 1.0 0.8 Fraction Bound 0.6 0.4 0.2 Data: Data1_B Model: XenoEstrogen 0.0

Chi^2/DoF = 0.04609 R^2 = 0.69397 -0.2 Kd 0.17725 0.06854 -0.4 -2 0 2 4 6

8 10 Free XenoEstrogen (uM) 12 14 16 Data Obtained Can answer the questions: How tightly does a certain xenoestrogen bind? How fast does it bind? Can estrogen compete off the xenoestrogen? Other chemically important information

Conclusions The Fluorescence Method for xeno-estrogen detection Quick & Highly sensitive Detect novel estrogen mimics in samples Obtain important biophysical data Questions SAR Questions OH 12 17 11 1 6 2 13

10 9 HO 3 4 5 16 8 7 14 15 Detection limit question (QCM) The binding of a small ligand

MW =< 500 closely packed protein protein diameter (~5-7 nm) 1:1 stoichiometry Yield a Change of 1-2 ng cm-2 or less Calculation with the Sauerbrey equation for a 10 MHz crystal yields an observed frequency change no greater than 0.4 Hz

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