ISO/TS 23459:2021
(Main)Nanotechnologies — Assessment of protein secondary structure during an interaction with nanomaterials using ultraviolet circular dichroism
Nanotechnologies — Assessment of protein secondary structure during an interaction with nanomaterials using ultraviolet circular dichroism
This document specifies measurement protocols and test conditions to determine alterations to protein secondary structure induced by their interaction with nanomaterials using ultraviolet circular dichroism (UV-CD) spectroscopy. This document does not apply to the characterization of conformational changes of disordered proteins.
Nanotechnologies — Évaluation de la structure secondaire des protéines durant une interaction avec des nanomatériaux à l'aide du dichroïsme circulaire ultraviolet
General Information
Standards Content (Sample)
TECHNICAL ISO/TS
SPECIFICATION 23459
First edition
2021-01
Nanotechnologies — Assessment of
protein secondary structure during an
interaction with nanomaterials using
ultraviolet circular dichroism
Nanotechnologies — Évaluation de la structure secondaire des
protéines durant une interaction avec des nanomatériaux à l'aide du
dichroïsme circulaire ultraviolet
Reference number
©
ISO 2021
© ISO 2021
All rights reserved. Unless otherwise specified, or required in the context of its implementation, no part of this publication may
be reproduced or utilized otherwise in any form or by any means, electronic or mechanical, including photocopying, or posting
on the internet or an intranet, without prior written permission. Permission can be requested from either ISO at the address
below or ISO’s member body in the country of the requester.
ISO copyright office
CP 401 • Ch. de Blandonnet 8
CH-1214 Vernier, Geneva
Phone: +41 22 749 01 11
Email: copyright@iso.org
Website: www.iso.org
Published in Switzerland
ii © ISO 2021 – All rights reserved
Contents Page
Foreword .iv
Introduction .v
1 Scope . 1
2 Normative references . 1
3 Terms and definitions . 1
4 Abbreviated terms . 2
5 Nanomaterial protein interactions . 2
6 Sample preparation . 3
6.1 General . 3
6.2 Desired properties of the UV-CD quartz cell. 3
6.3 Preparation of protein solution . 3
6.4 Instrumental setting condition . 4
6.5 Recording UV-CD spectra procedure. 4
6.5.1 General. 4
6.5.2 Buffer . 4
6.5.3 Protein sample . 4
6.5.4 Stability of NP suspension in the protein solution . 5
6.6 Preparation of protein-NPs conjugated suspension . 5
6.7 UV-CD spectra measurement . 5
6.8 Calculation of molar ellipticity . 6
6.9 Data analysis . 6
7 Test report . 7
Annex A (informative) Typical UV-CD spectra of proteins . 8
Annex B (informative) Literature survey on structural changes of NOAA and proteins .9
Annex C (informative) Description of buffers that can be used for protein solubility .15
Annex D (informative) Unit conversions in CD measurements .19
Annex E (informative) Calculating the concentration range of the sample .20
Annex F (informative) Methods for estimation of secondary structures of protein .21
Annex G (informative) Typical data of UV-CD used for estimation of secondary structures of
protein .22
Bibliography .23
Foreword
ISO (the International Organization for Standardization) is a worldwide federation of national standards
bodies (ISO member bodies). The work of preparing International Standards is normally carried out
through ISO technical committees. Each member body interested in a subject for which a technical
committee has been established has the right to be represented on that committee. International
organizations, governmental and non-governmental, in liaison with ISO, also take part in the work.
ISO collaborates closely with the International Electrotechnical Commission (IEC) on all matters of
electrotechnical standardization.
The procedures used to develop this document and those intended for its further maintenance are
described in the ISO/IEC Directives, Part 1. In particular, the different approval criteria needed for the
different types of ISO documents should be noted. This document was drafted in accordance with the
editorial rules of the ISO/IEC Directives, Part 2 (see www .iso .org/ directives).
Attention is drawn to the possibility that some of the elements of this document may be the subject of
patent rights. ISO shall not be held responsible for identifying any or all such patent rights. Details of
any patent rights identified during the development of the document will be in the Introduction and/or
on the ISO list of patent declarations received (see www .iso .org/ patents).
Any trade name used in this document is information given for the convenience of users and does not
constitute an endorsement.
For an explanation of the voluntary nature of standards, the meaning of ISO specific terms and
expressions related to conformity assessment, as well as information about ISO’s adherence to the
World Trade Organization (WTO) principles in the Technical Barriers to Trade (TBT), see www .iso .org/
iso/ foreword .html.
This document was prepared by Technical Committee ISO/TC 229, Nanotechnologies.
Any feedback or questions on this document should be directed to the user’s national standards body. A
complete listing of these bodies can be found at www .iso .org/ members .html.
iv © ISO 2021 – All rights reserved
Introduction
Nano-objects and their aggregates and agglomerates (NOAA) are currently produced in large mass
quantities globally and used in a variety of applications. However, there is concern about their
interaction with biological systems, including proteins, which could lead to reversible or irreversible
alterations in their secondary structure. The latter could affect the functionality and conformation
of protein, which in turn might affect the overall bio-reactivity of the proteins. The monitoring of the
occurrence of such alterations could thus provide important information on the interaction of NOAAs
with biological systems.
The process of folding of polypeptides in biological media produces the secondary structure of proteins
which determines their bioactivity. The important features of this structure include hydrogen bonds
between the amine hydrogen and carbonyl oxygen atoms in the peptide backbone and disulfide bonds
between two cysteine residues.
The protein secondary structure could be affected by exposing it to certain metallic ions and bioactive
compounds. Furthermore, it is also influenced by different buffer ionic strength, pH values, and
[1]
temperature . Alterations in the functionality and conformation of proteins can be attributed to
reorganization (so-called misfolding) and changes of the overall molecular dimension that accompany
the folding process. Some diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s and
[2]
Parkinson’s, are a consequence of misfolded proteins .
There are several standard techniques for determining the molecular structures/conformations and
folding process of proteins and upon their interaction with NOAAs. These include high-field nuclear
magnetic resonance (NMR), Fourier-transform infrared (FT-IR), Raman spectroscopy and ultraviolet
[3][4][5][6]
circular dichroism (UV-CD) spectroscopies . In addition, a novel technique synchrotron
radiation circular dichroism (SRCD) spectroscopy is a sensitive method to provide information on
[7]
protein secondary structures and folding .
TECHNICAL SPECIFICATION ISO/TS 23459:2021(E)
Nanotechnologies — Assessment of protein secondary
structure during an interaction with nanomaterials using
ultraviolet circular dichroism
1 Scope
This document specifies measurement protocols and test conditions to determine alterations to
protein secondary structure induced by their interaction with nanomaterials using ultraviolet circular
dichroism (UV-CD) spectroscopy.
This document does not apply to the characterization of conformational changes of disordered proteins.
2 Normative references
The following documents are referred to in the text in such a way that some or all of their content
constitutes requirements of this document. For dated references, only the edition cited applies. For
undated references, the latest edition of the referenced document (including any amendments) applies.
ISO/TS 80004-1, Nanotechnologies — Vocabulary — Part 1: Core terms
ISO/TS 80004-2, Nanotechnologies — Vocabulary — Part 2: Nano-objects
ISO/TS 80004-4, Nanotechnologies — Vocabulary — Part 4: Nanostructured materials
ISO/TS 80004-6, Nanotechnologies — Vocabulary — Part 6: Nano-object characterization
3 Terms and definitions
For the purposes of this document, the terms and definitions given in ISO/TS 80004-1, ISO/TS 80004-2,
ISO/TS 80004-4, ISO/TS 80004-6 and the following apply.
ISO and IEC maintain terminological databases for use in standardization at the following addresses:
— ISO Online browsing platform: available at https:// www .iso .org/ obp
— IEC Electropedia: available at http:// www .electropedia .org/
3.1
nanoparticle
NP
nano-object with all external dimensions in the nanoscale where the lengths of the longest and the
shortest axes of the nano-object do not differ significantly
Note 1 to entry: If the dimensions differ significantly (typically by more than three times), terms such as
“nanofibre” or “nanoplate” may be preferred to the term “nanoparticle”.
[SOURCE: ISO/TS 80004-2:2015, 4.4]
3.2
nanomaterial
material with any external dimension in the nanoscale or having internal structure or surface structure
in the nanoscale
Note 1 to entry: This generic term is inclusive of nano-object and nanostructured material.
Note 2 to entry: See also “engineered nanomaterial”, “manufactured nanomaterial” and “incidental nanomaterial”.
[SOURCE: ISO/TS 80004-1:2015, 2.4]
3.3
circular dichroism
optical effect of the differential absorption of left- and right-handed circularly polarized light
Note 1 to entry: Ultraviolet circular dichroism spectroscopy is used to investigate the secondary structure of
proteins.
4 Abbreviated terms
Ag-NP silver nanoparticle
Au-NP gold nanoparticle
AU absorbance unit
BSA bovine serum albumin
CD circular dichroism
DLS dynamic light scattering
HSA human serum albumin
MRE mean residue ellipticity
MWCNT multiwall carbon nanotubes
NOAA nano-objects and their aggregates and agglomerates
PAA-GNP poly (acrylic acid)-coated gold nanoparticles
SRCD synchrotron radiation circular dichroism
SWCNT single-wall carbon nanotubes
UV-CD ultraviolet circular dichroism
UV-Vis ultraviolet-visible
5 Nanomaterial protein interactions
In a biological environment, NOAA can easily interact with proteins such as apolipoproteins, fibronectin,
[5]
human serum albumin (HSA), vitronectin, etc. The layers of bound or adsorbed proteins around
[8]
NOAAs are called protein corona . Physicochemical characteristics of the nanomaterials (i.e. size,
surface area, hydrophobicity, charge density, surface chemistry, morphology) could affect the interaction
with surrounding biological compounds. The possible interaction between nanomaterials and these
compounds depends on protein association and dissociation kinetics. Nanomaterial–ligand complexes
[3][4][5][6][7][8][9]
have a lifespan ranging from microseconds to days . Nanomaterial-protein interaction
could lead to reversible or irreversible conformational changes on their secondary structures. Slight
changes in the secondary structure of proteins following the interaction with nanomaterials are
potentially reversible, whereas substantial changes could be irreversible. These changes can be
[9]
monitored by recording UV-CD spectra . UV-CD spectroscopy has its origin in the photophysical
process by raising an electron from ground state to an electronically excited state. UV-CD spectroscopy
is extensively used in the characterization of secondary structure, folding and binding properties of
[4][5][6]
proteins . The technique uses polarized light and measures the difference in absorbance between
the left- and right-handed circularly polarized light result in a UV-CD signal. Absorptions below 240 nm
2 © ISO 2021 – All rights reserved
are due to peptide bonds and absorptions in the range of 260 nm to 320 nm are due to aromatic amino
acid side chains. α-Helix, β-sheet, and β-turns are the most common secondary structural elements (see
Annex A and Figure C.1). It should be noted that aromatic amino acid side chains can also contribute to
the CD spectrum below 240 nm. It should also be noted that disulfide bonds can contribute to the CD
spectrum in both wavelength regions. Protein tertiary structure characterization is beyond the scope
of this document.
The structural alteration of critical human proteins following interaction with NPs has been reported
using UV-CD (see Table B.1). For instance, the irreversible structural changes caused by interaction of
human transferrin with superparamagnetic iron oxide NP (SPION), and fibrinogen with Au-NPs, led
[10]
to the loss of their primary biological function . The role of physical force on NP-cell interactions
investigated by studying interactions between Ag-NPs and HSA using UV-Vis, transmission electron
microscopy (TEM) and UV-CD measurement methods. It has been revealed that Ag-NPs-HSA binding
is mediated by hydrogen bonding, electrostatic and hydrophobic interactions that causes α-helices
[11]
decrease, and β-sheets increase, thereby changing protein biological function . The interaction
and stability of HSA-AgNP has been studied by SRCD spectroscopy and results showed reduction of
[12][13]
α-helix content of protein structure . PAA-GNP binding produced misfolding of Mac-1 protein,
which promotes interaction with the integrin receptor. Activation of this receptor increase the NF-kB
[14]
signalling pathway, resulting in the release of inflammatory cytokines .
6 Sample preparation
6.1 General
For recording spectra, an UV-CD instrument is needed with a data-acquiring range from 175 nm to
700 nm with a temperature control unit. A quartz glass cell (either rectangular or cylindrical) with
path lengths ranging from 0,5 mm to 10,0 mm is required. For recording UV-CD spectra, all material,
solvents and buffers should have low absorption in UV range. They should be as transparent as possible.
Working with optically active buffers creates additional challenges and is not recommended (see
Tables C.1, C.2 and C.3). For handling the proteins, special functionalized glassware with low-binding
affinity to protein should be used. All solutions shall be prepared with deionized water.
6.2 Desired properties of the UV-CD quartz cell
The UV-CD spectra should be recorded in highly transparent quartz cells. The cells shall have no optical
activity and desired path lengths ranging from 0,5 mm to 10,0 mm (rectangular or cylindrical). The
cells shall be thoroughly cleaned between the individual measurements (see Annex C).
6.3 Preparation of protein solution
Use a low-surface protein affinity test tube to weigh the protein and add a buffer solution to make a
stock solution with a suitable concentration. The required concentration can be determined using molar
[11]
extinction coefficients by the spectrophotometric method . The buffer shall be chosen according to
the type of protein and the type of NP used in the study. Prepare a stock solution of protein in the
concentration range of 1,0 mg/ml to 5,0 mg/ml. The stock solution can then be diluted for the UV-CD
measurements. The UV-CD spectra of proteins are recorded in 0,5 mm to 10,0 mm cells, a concentration
of 0,005 mg/ml to 5,0 mg/ml depending on the path length and the type of buffer. An acceptable UV-
CD spectrum should be obtained with desired protein contents between 0,005 mg/ml to 0,300 mg/ml
depending on the cell path length. For typical UV-CD measurements:
— in a 0,5 mm cell: 0,2 mg/ml to 1,0 mg/ml protein and required volume 0,025 ml to 0,050 ml;
— in a 1,0 mm cell: 0,05 mg/ml to 0,2 mg/ml protein and required volume 0,3 ml to 0,4 ml;
— in a 2,0 mm cell: 0,1 mg/ml to 0,3 mg/ml protein and required volume 0,9 ml to 1,0 ml;
— in a 10,0 mm cell: 0,005 mg/ml to 0,01 mg/ml protein and required volume 3,0 ml to 4,0 ml.
The protein should produce a sufficient UV and UV-CD signal. The desired UV level for protein solutions
at the wavelength and path-length of interest should range from 0,5 AU to 1,0 AU. The optimum
absorbance level is 0,89 AU (see Figure C.2).
6.4 Instrumental setting condition
The equipment needs to be purged with nitrogen gas about 30 min to 60 min before starting the machine
(manufacturer’s suggested time). A water circulation bath is required for controlling the temperature
of analyses using a water-jacketed cell holder/software-controllable Peltier. The bath should be set
at the desired temperature, which shall be constant throughout the experiment. The lamp should be
turned on before experiments and allowed to stabilize the output (30 min to 40 min).
6.5 Recording UV-CD spectra procedure
6.5.1 General
[3][4]
Before starting UV-CD measurements, set the temperature to the desired value (25 °C) . To obtain
proper signal-to-noise ratio to adequate spectral resolution, set the bandwidth between 1,0 nm and
1,5 nm. The wavelength range adjustment depends on the sample and cell used:
— from 190 nm to 260 nm for 0,2 mg/ml to 0,8 mg/ml protein samples in a 0,1 mm cell;
— from 190 nm to 260 nm for 0,1 mg/ml to 0,2 mg/ml protein samples in 1,0 mm and 2,0 mm cells;
— from 190 nm to 260 nm for 0,01 mg/ml to 0,02 mg/ml protein samples in a 10,0 mm cell.
Data collection interval of 1,0 nm sets should be used for samples with low ellipticity and with a signal
to noise ratio of 0,10 nm to 0,25 nm. The recommended interval for measurement of UV-CD spectrum
ranges from 190 nm to 260 nm. Data should be collected at one nm per s.
6.5.2 Buffer
Record the spectrum of the buffer to make sure it does not have any ellipticity. Make sure that measuring
parameters such as slit width, scanning step, integration time and scanning speed/integration time are
the same as those that will be used for measuring the samples. The presence of any buffer-related UV-CD
effect by overlapping on the protein UV-CD could lead to a misleading result. The increases absorbance
of the buffer in comparison with deionized water will decrease the signal-to-noise ratio. The spectrum
of the buffer and deionized water should overlay each other, within the experimental error, but the
spectrum of the buffer usually has a lower signal-to-noise ratio than the spectrum of deionized water at
[4]
low wavelengths .
— The recommended buffer for dissolving protein is sodium or potassium phosphate with an optimal
concentration of 10 mM, which is used as blank sample.
— Avoid using buffers with interfering with UV-CD spectrum such as citrates, MOPS (3-(N-morpholino)
propanesulfonic acid), imidazole and dithiothreitol (DTT). The list of buffers and UV cut off are
presented in Table C.3.
— Record a CD spectrum of the buffer alone before starting with a sample. The obtained spectrum of
blank should be relatively flat to ensure the buffer absorbance is not a concern.
6.5.3 Protein sample
After cleaning the cell, it is filled with protein solution and UV-CD spectra are recorded. Repeat the
measurement five to six times. Overlay each spectrum and average the data sets. Smooth the spectra
[4]
of the sample and blank . A number of approaches are available for spectral smoothing. Typically, the
Savitsky-Golay smoothing algorithm with polynomial order of 3 and smoothing window of 20 pts is
4 © ISO 2021 – All rights reserved
[4] to [15]
used. Subtract the smoothed baseline from the smoothed spectrum of the sample . The ellipticity
for most proteins should be close to zero between 250 nm and 260 nm.
NOTE The data are inspected to avoid the introduction of distortion in the pre- and post-smoothing process.
6.5.4 Stability of NP suspension in the protein solution
The stability of NPs in the buffer used for dissolving protein needs to be tested to avoid any
agglomeration of NPs suspension. Ideally the agglomeration state should be the same in sample and
control. However, it is noted that many studies have shown that proteins enhance particle agglomeration
and thus this might not always be practical. Preliminary studies addressing NP agglomeration should
be undertaken prior to UV-CD analysis as part of experimental design, as these can inform decisions on
the choice of buffers and particle concentrations. The measurement can be achieved by DLS techniques
[16]
(see Reference [15] and ISO 22412 .
6.6 Preparation of protein-NPs conjugated suspension
Prepare the protein-NPs conjugated suspension as follows:
a) Use glassware with low affinity to proteins.
b) Pipette the pre-calculated amount of protein stock solution into each vial at the same concentration.
c) Fill the glassware with sufficient amount of water to have a constant protein concentration.
d) Gently shake the vials and incubate them for about 5 min at room temperature (25 °C).
e) Add the fixed volume of nanomaterials suspension (10 µg/ml to 100 µg/ml) to the protein solution
of constant concentration, followed by gentle mixing (the correct ratio of protein and NPs can be
found in Annex E).
f) Incubate the prepared samples for 4 h at room temperature (25 °C).
g) Transfer the solution to the UV-CD cells.
h) Record spectra using a UV-CD cell over a range of 190 nm to 260 nm at room temperature (25 °C).
Collect data at 1,0 nm with a bandwidth of 1 nm, at 50 nm/min and averaging over five to six scans.
The final spectra should be baseline-corrected and data presented as mean residue ellipticity
(MRE), which is explained in 6.8.
6.7 UV-CD spectra measurement
Measure the UV-CD spectra as follows:
a) Record a UV-CD spectrum of the desired buffer (see Tables C.1 and C.2).
b) It is possible that reagents remain in the NP dispersion. Record UV-CD spectra of the corresponding
solution.
c) Test the UV absorbance of the used NPs in the range of 190 nm to 260 nm. If the absorbance of
the NP in this region is greater than 1,0 AU, the particle is not suitable for UV-CD experiment (see
Figures C.1 and C.2).
d) Carry out UV-CD measurements with the incubated protein–NP dispersion for at least five to six
replicates to test the reproducibility. If the reproducibility is not acceptable, it can point to an
insufficient equilibration time or a destabilized suspension.
e) Subtract the background/blank spectrum from sample data.
The CD spectra of NOAA in the range of 190 nm to 260 nm shall be recorded. The achiral NOAA shows
no CD effect in range of interest (see C.3). The NOAA which show a strong CD effect will not be suitable
for this type of investigation. All procedures for the recording of CD experiment shall be identical for
the NOAA, blank and protein sample.
6.8 Calculation of molar ellipticity
Quantitative analysis of the α-helix content in the protein can be calculated by converting the UV-CD
signal (see Figure C.2 and Table D.1) to the MRE using Formula (1):
θ
[]θ = (1)
10Cnl
where
[θ] is the MRE at 222 nm;
θ is the observed ellipticity in mdeg;
C is the molar concentration of the protein;
n is the number of amino acid residues;
l is the path length in cm.
The percentage of helicity is calculated using Formula (2):
(3θ − 000)
[]
H= (2)
(3−−6000 3000)
where
H is the α-helix (%);
[θ] is the observed MRE at 222 nm;
3 000 is the MRE [θ] of the random coil and β-form conformation cross at 222 nm;
36 000 is the MRE [θ] value of pure α-helix at 222 nm.
6.9 Data analysis
The estimation of protein secondary structure is carried out by quantitative analysis of UV-CD spectra.
Many validated reference spectra are publicly available from the Protein Circular Dichroism Data
[17]
Bank (PCDDB) . The deconvolution of UV-CD spectra can be carried out via different methods (see
Table F.1). There are a number of platforms which can be used for the estimation proteins structural
[18] [19] [20]
contents, such as CDPro , ValiDichro , PDB2CD, K2D3, DichroCalc, DICHROWEB, CCA+ and Beta
[21][22]
Structure Selection (BeStSel) . There are also other simplified procedures for the determination
[4][23][24][25]
of the structural content. . By applying such methods, the secondary structure of proteins
can be estimated and the changes made to the protein’s three dimensional structure as a result of
interactions with NPs can be measured. The changes above 5 % in the secondary structure of the
[57]
proteins are significant and ≥ 90 % on secondary structure of proteins will be denatured .
The measured UV-CD spectra can be transferred to the web server as a text file (see Annex G) or it can
be copied into the window in two data columns: data in units of either delta epsilon, MRE or mdeg. The
wavelength interval for input data at intervals of 1,0 nm is recommended. The output of structural
content estimation is obtained as a graphical presentation superposition of experimental and estimated
spectra, the listing of the secondary structure composition, total content and the spectral fitting with
root mean square deviation (RMSD) and normalized RMSD (NRMSD) data. It can be saved either in the
graphical form or in text format for further data processing or figure preparation. The users can adjust
6 © ISO 2021 – All rights reserved
the wavelength range and use a scaling factor to recalculate the results. The form of the output data can
be modified for the convenience of the user.
7 Test report
The test report shall include the information given in Table 1.
Table 1 — Test report
General
Product name: Product application:
Batch no.: Manufacturing method:
Lot no.:
Lab. name: Lab. address:
NOAA characteristics
Size Chemical composition
Shape Surface chemistry
a
NOAA concentration (M)
Protein characteristics
Protein concentration (mg/ml) pH
Estimated secondary structure content
The protein before interaction with The protein after interaction with
Structural content
NOAA (%) NOAA (%)
Helix
Antiparallel
Parallel
Others
RMSD
NRMSD
Type of database used for protein structure content analysis:
a
The concentration is expressed in µg/ml.
Annex A
(informative)
Typical UV-CD spectra of proteins
a) The various secondary structure of b) Represent
...
Questions, Comments and Discussion
Ask us and Technical Secretary will try to provide an answer. You can facilitate discussion about the standard in here.
Loading comments...