Analytical asse peptide-metal interactio Analytical assessment of metal interactions and ...

337 Pages·2010·5.38 MB·English

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stability

Laura A. Byrne B.Sc. (Hons)

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ffoorr tthhee ddeeggrreeee ooff Doctor of Philosophy (Ph.D.)

May 2010

RReesseeaarrcchh SSuuppeerrvviissoorrss:: Dr. Richard Murphy
Dr. Cathal Connolly
Prof. Sean Doyle

HHeeaadd ooff DDeeppaarrttmmeenntt:: Prof. Kay Ohlendieck

Department of Biology
Faculty of Science
National University of Ireland
Maynooth,
Co. Kildare,
Ireland

I
Table of Contents
List of Figures V
List of Tables XIII
Declaration of Authorship XVI
Dedication XVII
Acknowledgements XVIII
Abstract XIX
Abbreviations XX
1. Introduction ........................................................................................... 1
1.1 General introduction.............................................................................................. 1
1.2 Trace minerals in animal nutrition ........................................................................ 5
1.3 Bioavailability ....................................................................................................... 7
1.4 Inorganic versus organic trace minerals ................................................................ 9
1.4.1 Ligand sources ............................................................................................ 10
1.5 Complexes and chelates ...................................................................................... 14
1.6 Methods of comparative analysis for metal proteinates ...................................... 22
1.6.1 Surface Enhanced Laser Desorption/Ionisation Time of Flight Mass
Spectrometry (SELDI-ToF-MS) ................................................................. 26
1.6.2 Tandem Mass Spectrometry (MS/MS) ....................................................... 31
1.6.3 Enzyme-Linked Immunosorbent Assay (ELISA) ....................................... 32
1.6.4 Introduction to stability constants ............................................................... 34
1.6.5 Potentiometry .............................................................................................. 39
1.6.6 WinCOMICS and Hyperquad ..................................................................... 43
1.7 Aims and objectives of the study ........................................................................ 45
2. Materials and Methods ....................................................................... 47
2.1 Reagents and equipment ..................................................................................... 47
2.2 Laboratory preparation and analysis of metal proteinates .................................. 48
2.2.1 Hydrolysis of a protein source .................................................................... 48
2.2.2 Formation of metal proteinates from soy flour hydrolysate ....................... 48
I
2.2.3 Determination of total nitrogen ................................................................... 49
2.2.4 Free α-Amino Nitrogen (FAN) determination ............................................ 49
2.2.5 CHN analysis .............................................................................................. 50
2.2.6 Flame Atomic Absorption Spectroscopy .................................................... 50
2.2.7 Fourier Transform Infrared (FTIR) Spectroscopy ...................................... 51
2.2.8 Amino acid profiling ................................................................................... 51
2.2.9 Preparation of a metal proteinate mimic ..................................................... 51
2.3 Mass spectrometry .............................................................................................. 52
2.3.1 Calibration of Surface Enhanced Laser Desorption / Ionisation Time-of-
Flight Mass Spectrometer (SELDI-ToF-MS) Instrument ........................... 52
2.3.2 Analysis of suitable array surfaces and buffers for SELDI-ToF-MS ......... 54
2.3.3 SELDI-ToF-MS analysis of metal proteinates ............................................ 56
2.3.4 Tandem MS Collision-Activated Dissociation ........................................... 56
2.3.5 Premix analysis ........................................................................................... 57
2.3.6 Feed analysis ............................................................................................... 57
2.3.7 Synthetic peptide production ...................................................................... 57
2.4 Enzyme Linked Immunosorbent Assay (ELISA) ............................................... 58
2.5 Stability constant determination using potentiometry and ion-selective electrodes
(ISE) .................................................................................................................... 59
3. Results and Discussion ........................................................................ 62
3.1 Assessment of potential protein sources for metal proteinate production .......... 62
3.1.1 Total nitrogen analysis of protein sources .................................................. 64
3.1.2 Hydrolysis of protein sources...................................................................... 65
3.1.3 Optimisation of hydrolysis parameters ....................................................... 67
3.1.3.1 Optimisation of hydrolysis temperature .............................................. 69
3.1.3.2 Optimisation of hydrolysis pH ............................................................ 72
3.1.3.3 Optimisation of enzyme concentration ............................................... 73
3.2 Metal proteinate production ................................................................................ 77
3.2.1 Formation of metal proteinates ................................................................... 77
3.3 Metal proteinate characterisation ........................................................................ 79
II
3.3.1 FAN analysis of metal proteinates .............................................................. 80
3.3.2 Determination of total metal content using flame Atomic Absorption
Spectrometry ............................................................................................... 81
3.3.3 CHN analysis .............................................................................................. 82
3.3.4 Fourier Transform InfraRed (FTIR) spectroscopy ...................................... 84
3.3.5 Amino acid profiling ................................................................................... 88
3.3.6 Surface-Enhanced-Laser-Desorption/Ionisation-Time-of-Flight-Mass-
Spectrometry (SELDI-ToF-MS) analysis of metal proteinates .................. 91
3.3.6.1 Calibration of SELDI-ToF-MS Instrument ......................................... 92
3.3.6.2 Selection of suitable array surfaces ..................................................... 93
3.3.6.3 Analysis of hydrolysed soy using Immobilised Metal Affinity Capture
(IMAC) arrays ................................................................................... 100
3.3.6.4 SELDI-ToF-MS analysis of a metal-peptide mimic ......................... 101
3.3.6.5 Immobilised Metal Affinity Capture (IMAC) analysis of metal
proteinates ......................................................................................... 104
3.3.6.6 Marker peptide identification ............................................................ 119
3.3.6.7 Premix analysis ................................................................................. 121
3.3.6.8 Feed analysis ..................................................................................... 125
3.3.6.9 Tandem Mass Spectrometry (MS/MS) ............................................. 129
3.4 Synthetic peptide analysis ................................................................................. 134
3.5 Antibody assay .................................................................................................. 135
3.6 The use of potentiometry and ISE titrations to determine stability constants... 140
3.6.1 Calibration of the Cu(II) ion selective electrode ....................................... 143
3.6.2 Potentiometric titrations of amino acids and Cu2+ using an ISE. .............. 145
3.6.3 Potentiometric titrations of peptide ligands and Cu2+ using an ISE .......... 154
3.6.4 Potentiometric titrations of polypeptides and Cu2+ using an ISE. ............ 175
3.6.5 Potentiometric titrations of novel peptide ligands and Cu2+ using an ISE 181
3.6.5.1 Determination of stability constants for the 1148 Da marker peptide with
Cu2+ ....................................................................................................... 182
3.6.5.2 Determination of stability constants for the 1181 Da marker peptide with
Cu2+ ....................................................................................................... 188
III
3.6.5.3 Determination of stability constants for the 1300 Da marker peptide with
Cu2+ ....................................................................................................... 192
3.6.5.4 Determination of stability constants for the 1514 Da marker peptide with
Cu2+ ....................................................................................................... 197
3.6.5.5 Identification of species proposed by Hyperquad in metal proteinates
using SELDI-ToF-MS ........................................................................... 202
3.6.6 Further applications of ISE electrodes in this work .................................. 207
3.6.6.1 Potentiometric titrations of hydrolysed soy and Cu2+ using an ISE .. 207
3.6.6.2 Effect of ligand hydrolysis on proteinate stability ............................ 209
3.6.6.3 Potentiometric titrations of metal proteinate test samples. ............... 263
4. Conclusion .......................................................................................... 269
Bibliography .................................................................................................. 274
Appendix 1 - FTIR ........................................................................................................ 291
Appendix 2 – Amino acids............................................................................................ 312

IV
List of Figures

Chapter 1. Introduction
Figure 1.1 Effect of declining trace mineral status on animal performance 3
Figure 1.2 General schematic of an amino acid 10
Figure 1.3 Stereochemistry of the peptide bond 11
Figure 1.4 Schematic diagrams of a metal chelate and a metal complex 15
Figure 1.5 Selection of amino acid side-chain metal-ion binding modes 17
Figure 1.6 Chelate formed from Copper and the tripeptide Gly-Gly-His 18
Figure 1.7 SELDI-ToF-MS chromatographic surface types 27
Figure 1.8 Schematic diagram of a Triple Quadrupole Time of Flight (QqToF)
mass spectrometer used to obtain peptide sequences 31
Figure 1.9 An example of a typical ELISA assay 33
Figure 1.10 Schematic of a combination ion-selective electrode 41

Chapter 3. Results and discussion
Figure 3.1 Schematic diagram of a 1:2, copper:glycine chelate 63
Figure 3.2 Proteolysis of peptide linkage
Figure 3.3 Free α-amino nitrogen (ppm) before and after hydrolysis 65
Figure 3.4 Effect of temperature adjustment on FAN release from soy flour 68
Figure 3.5 Assessment of thermal effects on enzymatic hydrolysates using
SELDI-ToF-MS 71
Figure 3.6 Effect of pH adjustment on FAN release from soy flour 72
Figure 3.7 Effect of adjusting enzyme concentration on FAN release from
soy flour 74
Figure 3.8 Assessment of the effect of varying enzyme concentration on soy
hydrolysates using SELDI-ToF-MS 76
Figure 3.9 Colour comparisons of copper, iron, manganese and zinc proteinates 79
Figure 3.10 Free α-amino nitrogen analyses of metal proteinates 81
Figure 3.11 FTIR spectra of copper(II) proteinates and soy ligand controls 85
Figure 3.12 IR spectra of soil fulvic acid at pH 2 (blue) and pH 6 (red) 87
V
Figure 3.13 Amino acid profile of a soy ligand 89
Figure 3.14 Internal calibration spectra obtained using All-in-One peptide
mixture 93
Figure 3.15 Cu proteinate analyses on a selection of array surfaces 94
Figure 3.16 Fe proteinate analyses on a selection of array surfaces 95
Figure 3.17 Mn proteinate analyses on a selection of array surfaces 96
Figure 3.18 Zn proteinate analyses on a selection of array surfaces 97
Figure 3.19 Wide spectral range analysis of a Mn(II) proteinate using
an NP20 array 98
Figure 3.20 Diagrams illustrating the NTA surface structure (A) and the binding
of a metal ion M+ to the NTA group (B) 99
Figure 3.21 SELDI-ToF-MS spectra of a hydrolysed and unhydrolysed
soy sample 100
Figure 3.22 Fully deprotonated EDTA molecule illustrating metal (M)
coordination 102
Figure 3.23 SELDI-ToF-MS spectra of ranatensin before and after treatment with
EDTA 103
Figure 3.24 SELDI-ToF-MS spectra (0 Da – 5000 Da) of a selection of metal
proteinates and controls 105
Figure 3.25 SELDI-ToF-MS spectra (0 Da – 500 Da) of a selection of metal
proteinates and controls 106
Figure 3.26 SELDI-ToF-MS spectra (500 Da – 1000 Da) of a selection of metal
proteinates and controls 108
Figure 3.27 SELDI-ToF-MS spectra (700 Da – 950 Da) of copper and zinc
proteinates treated with EDTA 109
Figure 3.28 SELDI-ToF-MS spectra (1000 Da – 1500 Da) of a selection of metal
proteinates and controls 111
Figure 3.29 SELDI-ToF-MS spectra (1150 Da – 1370 Da) of a zinc(II) proteinate
treated with EDTA 113
Figure 3.30 SELDI-ToF-MS spectra (1500 Da – 2000 Da) of a selection of metal
proteinates and controls 114
VI
Figure 3.31 SELDI-ToF-MS spectra (1140 Da – 1700 Da) of a copper proteinate
and controls 115
Figure 3.32 SELDI-ToF-MS sample analysis of Cu proteinate batches
manufactured using four different batches of soy flour 117
Figure 3.33 SELDI-ToF-MS spectra of the peptides of interest in laboratory
manufactured metal proteinate samples 120
Figure 3.34 SELDI-ToF-MS spectra of copper proteinates manufactured on a
pilot scale 120
Figure 3.35 Assessment of SELDI-ToF-MS for qualitative detection of metal
proteinates in premix material 123
Figure 3.36 Assessment of SELDI-ToF-MS for qualitative detection of metal
proteinates in premix material at a range of inclusion levels 124
Figure 3.37 Assessment of SELDI-ToF-MS for qualitative detection of metal
proteinates in feed 126
Figure 3.38 Assessment of SELDI-ToF-MS for qualitative detection of metal
proteinates in premix material at low inclusion levels 128
Figure 3.39 Tandem MS spectra of a copper proteinate sample 130
Figure 3.40 Tandem MS spectra of the 1148 Da marker peptide 130
Figure 3.41 Tandem MS spectra of the 1181 Da marker peptide 131
Figure 3.42 Tandem MS spectra of the 1300 Da marker peptide 131
Figure 3.43 Tandem MS spectra of the 1514 Da marker peptide 132
Figure 3.44 Analysis of the 1300 Da synthetic peptide using SELDI-ToF-MS 134
Figure 3.45 Schematic diagrams outlining an ELISA method 136
Figure 3.46 Standard curve of antibodies 1E2(♦) and 1H5(■) raised against the
1300Da synthetic peptide 137
Figure 3.47 ELISA of synthetic and native antigen containing samples and an
unhydrolysed soy control 138
Figure 3.48 Calibration curve for a Cu2+ ISE with (CuNO ) .2.5H O at 25 °C and
3 2 2
0.1 M ionic strength 144
Figure 3.49 Comparative analysis of published and experimental log K values for
amino acid ligands with Cu2+ 147
VII
Figure 3.50 Schematic of the complex formed between Cu and glycine 148
Figure 3.51 Experimental titration curves for 10mM Cu + 20mM Glycine
at 25 °C and 0.1 M ionic strength illustrating the decrease in
free Cu with increasing pH 149
Figure 3.52 Speciation diagram created using WinCOMICS for Cu (0.001 M)
and Glycine (0.002 M) at 25 °C and 0.1 M ionic strength,
[M]:[L] = 1:2 150
Figure 3.53 Screenshot capture of the Hyperquad file for a Cu-Gly titration 151
Figure 3.54 FTIR spectra of a copper(II) sulphate pentahydrate control,
histidine and a His-Cu complex 153
Figure 3.55 The general formula for a dipeptides comprised of two amino acids 154
Figure 3.56 Screenshot capture of the Hyperquad file for a copper(II)-glygly
titration 156
Figure 3.57 Chelate formed from copper and the dipeptide Gly-His 157
Figure 3.58 Speciation diagram created using WinCOMICS for Cu (0.001 M)
and diglycine (0.002 M) at 25 °C and 0.1 M ionic strength,
[M]:[L] = 1:2 160
Figure 3.59 Screenshot capture of the Hyperquad file of diglycine with Cu2+ 160
Figure 3.60 FTIR spectra of a copper(II) sulphate pentahydrate control,
diglycine and a GlyGly-Cu complex 163
Figure 3.61 Speciation diagram created using WinCOMICS for Cu (0.001 M)
and triglycine (0.002 M) at 25 °C and 0.1 M ionic strength,
[M]:[L] = 1:2 164
Figure 3.62 Screenshot capture of the Hyperquad file of triglycine with Cu2+ 164
Figure 3.63 FTIR spectra of a copper(II) sulphate pentahydrate control,
triglycine and a (Gly) -Cu complex 167
3
Figure 3.64 Speciation diagram created using WinCOMICS for Cu (0.001 M)
and tetraglycine (0.002 M) at 25 °C and 0.1 M ionic strength,
[M]:[L] = 1:2 167
Figure 3.65 Screenshot capture of the Hyperquad file of tetraglycine with Cu2+ 168

VIII
Figure 3.66 FTIR spectra of a copper(II) sulphate pentahydrate control,
tetraglycine and a (Gly) -Cu complex 169
4
Figure 3.67 Speciation diagram created using WinCOMICS for Cu (0.001 M)
and pentaglycine (0.002 M) at 25 °C and 0.1 M ionic strength,
[M]:[L] = 1:2 170
Figure 3.68 Screenshot capture of the Hyperquad file of pentaglycine with Cu2+ 170
Figure 3.69 FTIR spectra of pentaglycine and a (Gly) -Cu complex 171
5
Figure 3.70 Structure of ranatensin, a polypeptide used as a metal peptide mimic 176
Figure 3.71 SELDI-ToF-MS spectra of ranatensin (Panel 1), the formation of a
ranatensin-Cu complex (Panel 2) and the complex after treatment
with EDTA (Panel 3) 178
Figure 3.72 Screenshot capture of the Hyperquad file of the reaction between
Cu2+ and ranatensin at pH 5.5 180
Figure 3.73 Screenshot capture of the Hyperquad file of the 1148 Da synthetic
peptide with Cu2+ at pH 5.5 and 0.1 M ionic strength at 25 oC 184
Figure 3.74 SELDI-ToF-MS spectra of the 1148 Da synthetic peptide before and after
titration with Cu and on treatment with EDTA 185
Figure 3.75 FTIR spectra of the 1148 Da synthetic peptide titrated with Cu(II)
and a copper and a peptide control 186
Figure 3.76 Structures of 1:1, metal:ligand complex species for (a) Gly-His
(CuH L), (b) Gly-Gly-His (CuH L) and (c) Gly-Gly-Gly-His 187
-1 -2
Figure 3.77 Screenshot capture of the Hyperquad file of the 1181 Da synthetic
peptide with Cu2+ at pH 5.5 and 0.1 M ionic strength at 25 oC 189
Figure 3.78 SELDI-ToF-MS spectra of the 1181 Da synthetic peptide before and after
titration with Cu 190
Figure 3.79 FTIR spectra of the 1181 Da synthetic peptide titrated with Cu(II)
and a copper and a peptide control 191
Figure 3.80 Screenshot capture of the Hyperquad file of the 1300 Da synthetic
peptide with Cu2+ at pH 5.5 and 0.1 M ionic strength at 25 oC 193
Figure 3.81 SELDI-ToF-MS spectra of the 1300 Da synthetic peptide before and after
titrating with Cu 194
IX