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Research ArticleReceived: 24 November 2008 Accepted: 1 March 2009 Published online in Wiley Interscience: 8 April 2009

(www.interscience.wiley.com) DOI 10.1002/jrs.2283

Real-time measurement of H/D exchange in amicrodialysis Raman quartz cellPedro Carmonaa∗ and Marina Molinab

We present here a novel quartz cell for monitoring H/D exchange in biomolecules using Raman spectroscopy. This cell iscombined with a syringe to pump heavy water through a hollow microdialysis fibre, which is inserted into the cell. Thedeuterium efflux into the sample has been studied as a function of the molecular weight cut-off of the microdialysis fibre andcompared with other microcell systems comprising conventional glass capillaries. The fastest D2O efflux that we have obtained(kd = 0.38 ± 0.008 min−1) permits to measure exchange rates of 2.5 min−1 or less. Application of this cell to deuteriumexchange in glyceraldehyde-3-phosphate dehydrogenase reveals a class of H-atoms highly resistant to deuteration, which isconsistent with a previous infrared study on this protein. Copyright c© 2009 John Wiley & Sons, Ltd.

Keywords: Raman spectroscopy; hydrogen-deuterium exchange; microdialysis cell; glyceraldehydes-3-phosphate dehydrogenase

Introduction

Hydrogen–isotope exchange has long been used for the analysis ofprotein and nucleic acid structures and dynamics.[1 – 3] Exchangingrates depend on pH, temperature and biomolecular environment.Groups exposed to the solvent exchange fastest, and thehydrogens of a structured region in the said biomoleculesexchange more slowly compared with the hydrogens of anunstructured part. This is because of hydrogen bonding, lowsolvent accessibility and steric blocking. In order to monitor thetime course of the isotope exchange, the lyophilised biomoleculein question dissolved in D2O can be observed in Ramanexperiments. However, one cannot measure the isotope exchangeoccurring from the moment the substance is dissolved until themoment spectral recording starts. In addition, unfortunately,the lyophilised state sometimes causes biomacromoleculesto aggregate, which involves undesired structural alterations.Another alternative method for monitoring the isotope exchangeprocess is dialysing the aqueous solution considered againstD2O buffer and taking samples from the dialysis tube in thecourse of the exchange process, which requires relatively largequantities of biological sample. These are the reasons why Ramanmicrodialysis cells that require only microlitre sample volumeswere used by other authors to investigate exchange kineticsof biological assemblies.[4,5] The sample solutions (∼10 µl) wereplaced in standard glass capillary tubes where an appropriatemicrodialysis tubing of 200 µm outer diameter was inserted.The method is based upon the measurement in real time ofthe Raman band intensities associated with specific moleculargroups of proteins and nucleic acids, as these groups becomeprogressively exchanged by either deuterium or protium in a D2Oor H2O solvent environment, respectively. On this experimentalbasis, it is expected that the more changes are resolved themore detailed structural information can be obtained. Thecorresponding time-resolved Raman spectra can be subsequentlyanalysed by two-dimensional correlation spectroscopy that offersthe very well-known advantages compared with conventionalmonodimensional spectroscopy.[6]

The intensity of Raman scattered light from a molecular speciesis proportional, among other factors, to the volume of sampleilluminated by the laser source and viewed by the spectrometer.[7]

This means that the use of sample volumes greater than thoseincluded in capillary tubes is advantageous in case of lowconcentration samples and/or exciting with a laser beam ofrelatively long wavelength to circumvent fluorescence. In thisconnection, it can be suitable that the use of 40 µl quartz cellsof 1 cm path length that, on the other hand, is usually employedfor spectral absorption measurements in the ultraviolet (UV)-visible range. To our knowledge, no study has been carried outabout a real-time measurement of the H/D exchange in the saidmicrodialysis quartz cells. Apart from the dependence of solventefflux rate on the type of capillary and quartz cell, we have alsoexamined the effects of the microdialysis tubing molecular cut-offand reported some protein and polynucleotide exchange profilesmeasured in the said Raman microdialysis quartz cell.

Experimental

Materials

For Raman spectroscopy, glyceraldehyde-3-phosphate dehydro-genase (GAPDH) was purchased from Sigma and used withoutfurther purification. The purity of this protein was checked by SDS-PAGE, and contaminating proteins were found in small amount(<2%). GAPDH solution was prepared at 3% w/w concentration ina 50 mM Tris buffer (pH 7.4). Polyribocytidylic acid [poly(rC)] wasacquired from Sigma and its aqueous solutions for spectroscopicmeasurements were prepared with 3% w/w concentration at pH7.4 in 50 mM Tris buffer containing 0.1 M NaCl.

∗ Correspondence to: Pedro Carmona, Instituto de Estructura de la Materia (CSIC),Serrano 121, 28006 Madrid, Spain. E-mail: p.carmona@iem.cfmac.csic.es

a Instituto de Estructura de la Materia (CSIC), 28006 Madrid, Spain

b Departamento de Química Organica I, Escuela Universitaria de Optica, 28037Madrid, Spain

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D2O for H/D isotopic exchange was acquired from Aldrich andits minimum isotopic purity was 99.9 atom % D. The constituentsof the above H2O and D2O buffers were free from RNAases andDNAases and purchased from Fluka.

Regenerated cellulose hollow fibres of 200 and 216 µm innerand outer diameter, respectively, were acquired from Spectra/Por(Spectrum, Spectrum Laboratories Inc.). They were used with 13and 18 kDa molecular weight cut-off (MWCO) for microdialysis.

Instrumentation

Two flow cells were designed for measurement of isotopicexchange. One of them (Fig. 1) consists of a syringe pump with aconical outlet sealed to a Spectra/Por microdialysis hollow fibre.The microdialysis fibre used is passed through a drilled cap of thequartz cell, so that approximately 8 mm length of fibre can beinserted into the liquid sample. The loop shape of the hollow fibreis achieved through the drilled Teflon cap of the quartz cell whoselower part contains two curved conductive tubes of about 0.5 and1 mm inner and outer diameter, respectively. These tubes can beof stainless steel or glassy nature. The upper part of the cell capcontains two inserted glass capillary tubes of about 10 mm lengthand 1 mm inner diameter, which are sealed at their ends with ageneric rosin wax in order to fix the microdialysis fibre. An end ofthis is connected to the syringe through its corresponding glasscapillary tube that is inserted into a silicone tube linked to theconical outlet of the syringe. Obviously, the inner diameter of thesilicone tubing must be slightly smaller than the outer diameter ofthe glass capillary tube in order to avoid leaks of heavy water andto make this deuterated solvent to pass along the microdialysisfibre. The shape of the sample volume in the cell is a tetragonalprism having a base side length of 2 mm and height of 10 mm. Aconstant flow of 3–4 ml/h is controlled by the syringe pump. Formonitoring isotopic exchange, 40 µl liquid sample is placed in thequartz cell with the aid of an adjustable micropipette containinga thin tip. The Teflon stopper having the microdialysis fibre isthen inserted into the quartz cell, so that about 8 mm length offibre is immersed in the upper part of the liquid sample. D2Oeffluent is then pumped through the dialysis fibre at the indicatedflow rate, while Raman spectra in the 1800–700 cm−1 range were

syringe pump

microdialysis fibre

laser

Figure 1. Scheme of the microdialysis quartz cell for real-time measure-ment of H/D exchange by Raman spectroscopy.

collected at 1 min intervals with 20 s data accumulation times.Thus, three spectra were captured for every 1-min interval andtheir signals were averaged to improve signal-to-noise ratios.The Raman bands appearing near 1205 and 1640 cm−1 becauseof the δD2O and δH2O vibrational modes, respectively, can beused to compare the exchange profile of solvent with that ofthe solute in question. In this connection, the intensity of theδD2O 1205 cm−1 band can be used for measuring the solventdeuteration when the solute in question generates Raman signalsthat overlap with the δH2O water band at 1640 cm−1, as occursfor proteins. In the case of GAPDH and in order to measure δD2Ointensities, the small interference of the 1208 cm−1 weak bandwas removed by subtracting the initial aqueous protein spectrumwith the use of the 1003 cm−1 phenylalanine band as internalstandard. The Raman spectra were recorded with a RenishawRaman System RM2000 equipped with the 785 nm laser line, anelectrically refrigerated CCD camera and a notch filter to eliminatethe elastic scattering. The output laser power was 5.0 mW and thespectral resolution was 4 cm−1. The Raman data were collected in180◦ backscattered geometry and were fed to a microcomputerfor storage, display, plotting and processing, and the manipulationand evaluation of the spectra were carried out using the Grams/AIsoftware (Thermogalactic). The spectra were smoothed with theSavitzky–Golay procedure on 11 points. Data processing was alsocarried out for multipoint baseline correction, and care was takento achieve a smooth baseline with no abrupt nicks.

The other microdialysis cell used here consists of a hollowfibre threaded through a conventional glass capillary tube of1.5 mm inner diameter, as described elsewhere.[5] Briefly, 10 µlliquid sample is placed in the glass capillary and then subjected toheavy water effluent through a hollow microdialysis fibre with aconstant flow of 3–4 ml/h controlled by the syringe pump. Ramanspectra were measured at the same conditions as those for theabove quartz cell.

Results and Discussion

The Raman measurement of the efflux of D2O into H2O for bothmicrodialysis cell types is shown in Fig. 2A. By analogy with thegeneral solution of the diffusion equation, the data expressed forevery time as a fraction of deuterium atoms in the aqueous solvent,�d(t), can be fitted to the equation[5]

�d(t) = 1 − exp(−kdt) (1)

yielding the kd efflux rate values of 0.36 ± 0.007 and 0.29 ±0.005 min−1 for capillary and quartz cell, respectively, usingmicrodialysis hollow fibre of 13 kDa MWCO (Table 1). The slightlylower kd value for the quartz cell compared with the capillarycell can be attributable to the greater path of molecular diffusionassociated with greater sample volume used in the quartz cell.[5]

The effect of dialysis fibre pore sizes on the D2O efflux rate isshown in Table 1 and Fig. 2B. As expected, greater pore sizesfavour effluent molecular diffusion, whereby kd increases withMWCO. Interestingly, kd is increased by a factor of about 1.3 ingoing from 13 to 18 kDa MWCO.

As an application of the microdialysis quartz cell to H/Dexchange in proteins, we have measured this process for GAPDHin aqueous solution. Fig. 3 shows the Raman spectra of GAPDH inaqueous solution after 120-min exposure to heavy water. The topspectrum exhibits amide I and amide III bands with intensity

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Table 1. Dependence of D2O efflux rate (kd min−1) on some cellcharacteristics

MWCO(Da)

Capillarycell

Quartzcell

18 000 0.48 ± 0.006 0.38 ± 0.008

13 000 0.36 ± 0.007 0.29 ± 0.005

6 000 0.28 ± 0.005a

a Reference [5].

0 5 10 15 20 25

0.0

0.2

0.4

0.6

0.8

1.0

D2O

effl

ux

Time/min

B

0 5 10 15 20 25

0.0

0.2

0.4

0.6

0.8

1.0

D2O

effl

ux

Time/min

A

Figure 2. Plots of deuterium exchange of H2O versus time of D2O efflux.(A) Comparison of glass capillary tube (•) (kd = 0.36 ± 0.007 min−1) andquartz cell (◦) (kd = 0.29 ± 0.005 min−1), using 13 kDa microdialysishollow fibre. (B) Comparison of 18 kDa (•) (kd = 0.38 ± 0.008 min−1) and13 kDa (◦) (kd = 0.29 ± 0.005 min−1) hollow fibres in the quartz cell.

maxima at 1664 and 1233 cm−1, respectively, indicating thepresence of β-sheet conformations,[8] which is consistent withcrystal structure.[9] The sharp band at 1003 cm−1 is because ofthe phenylalanine residues and can serve as a convenient internalintensity standard. The strong amide I band at 1664 cm−1 shiftsto 1659 cm−1 on deuteration for 120 min. The intensity overlap ofamide I and amide I′ bands precludes the use of these spectralprofiles to determine exchange kinetics. Therefore, we haveused a lower wavenumber region for suitable measurements ofmarkers of NH protein backbone exchange. In this connection, themost significant spectral change accompanying NH deuterationin protein backbone is the shift of the medium-intensityconformation markers in the 1350–1220 cm−1 range (amide IIImodes) to the 990–900 cm−1 range (amide III′) (Fig. 3). The

1600 1400 1200 1000 800

1003

Ram

an In

tens

ity

Wavenumber/cm-1

1003

166

4

1555 14

1214

49

1340

1233

1208

1127 10

32 980

954

849

830

1659

1555

1449

1406 13

40

1261

1231

1207

1125 10

32

954

847

828

Figure 3. Top: Raman spectrum of GAPDH at 3% w/w concentration in50 mM Tris buffer (pH 7.4). Bottom: Raman spectrum of the same proteinsolution after 120-min D2O efflux.

amide III mode represents mainly coupled νCN and in-planeδNH motions, whereas amide III′ is considered to be largely anin-plane δND vibration.[8,10,11] The amide III profile is relativelybroad and comprises major contributions from 1233 cm−1 (β-sheets) and 1263 cm−1 (α-helices). On deuteration, the complexamide III profile generates a broad amide III′ band with anintensity maximum at 954 cm−1 and two shoulders at 962 and935 cm−1, which may reflect the presence of various proteinsecondary structures. The 980 cm−1 band, which is present inthe undeuterated solution spectrum, disappears in the course ofdeuteration. This band can be ascribed to νCN motions in basicamino acids, such as arginine and lysine,[12,13] which constituteabout 10% of amino acid composition of GAPDH.[14] Interestingly,after 120-min deuterium exposure, two bands are visible at 1261and 1231 cm−1 (Fig. 3), which can be ascribed to α-helix andβ-sheet protein backbone arrangements, respectively.[8,10,11] Thecrystal structure of GAPDH[9] shows subunit–subunit contactsformed by antiparallel β-sheets. In addition, a hydrophobic pocketis also formed by insertion of a β-sheet between α-helices. Theordering of protein backbone in α-helix and β-sheet segmentsand the said contacts between these hydrogen-bonded structurescertainly favour retardation of the isotopic exchange, as describedbelow, and is consistent with the presence of the two amide IIIband components located at 1261 and 1231 cm−1 after 120-mindeuterium exposure. The area increasing of the amide III′ region inthe course of deuteration has been used for quantitative isotopicexchange analysis. With this aim, time-resolved Raman spectrawere collected in the 1750–700 cm−1 range to investigate theexchange kinetics of the peptide bonds. Concurrent time-resolvedmeasurements of the solvent νOD band (2800–2200 cm−1)confirm that the kd value is 0.29 ± 0.005 min−1 (Fig. 4). Theseresults show that a fraction of around 30% of the protein backboneNH groups is exchangeable at the same conditions that permitcomplete solvent exchange. The remaining NH unexchangedfraction (70%) may correspond approximately to the protons in α-helices and β-sheets of GAPDH. In fact, the whole protein tetrameris found to have 35% α-helices and 39% β-sheets.[9] In addition,in other work dealing with the hydrogen–isotope exchangebetween GAPDH and heavy water, it was reported that GAPDH has

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0 20 40 60 80 100 120

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n ex

chan

ged

Time/(min)

Figure 4. The time course of deuterium exchange of GAPDH backbone NHbonds (◦) and buffer H2O (•) measured with the use of the quartz cell and13 kDa microdialysis hollow fibre.

a class of hydrogen atoms with 50–60 h half-lives.[15] Isotopicexchange kinetics of α-helix and β-sheet protein secondarystructures are nearly 10−2 and 5 × 10−4 min−1, respectively,[16]

and consequently these structures can be kinetically distinguishedfrom that of the solvent. Solvent exchange of protein NH amidegroups has been investigated extensively, and some reviewshave been given.[1,17,18] In random-coiled polypeptides, theobserved exchange average rate uses to be approximately 60 s−1

corresponding to a half-life t1/2 of 12 ms.[19] However, for globularproteins, amide exchange rates are often orders of magnitudeslower than those of unordered protein backbones. Thus,exchange rates vary considerably between different proteins, fromnearly complete exchange within 3 h to more than 50% of peptideprotons being exchange resistant for at least several hours.[20 – 22]

Retardation of NH exchange in proteins is a result of theinvolvement of NH groups in hydrogen-bonding interactions.[1]

Accordingly, peptide NH exchanges require transient breaking ofpeptide group hydrogen bonds and, consequently, are stronglyinfluenced by the structure of the protein in question, its solventaccessibility and strength of hydrogen bonds. The above factors,hence, can explain the different isotopic exchange behaviours ofthe aqueous solvent and protein shown in Fig. 4.

We have also applied the above quartz cell to monitor theshift of deuterium exchange-sensitive bands of a synthetic polyri-bonucleotide, namely poly(rC). Deuteration of the amino groupin this polynucleotide generates a wavenumber downshifting ofthe 784 cm−1 cytosine band to 774 cm−1, as reflected in Fig. 5.The time-resolved spectra in this region generate a curve (Fig. 6)showing that the cytosine deuteration exchange follows closelythe D2O efflux, which means that the observed exchange rate ofthis polynucleotide is limited only by the time constant of thequartz cell system. In fact, exchange measurements by stop-flowUV spectroscopy on poly(rC) at 25 ◦C and pH 7 indicate a exchangerate (kC = 12.6 s−1), which significantly exceeds the upper limitof detection of the present experimental system.[23] In order toknow this limit, we have assumed that the deuteration process is ofsecond-order character. Unlike rapid mixing methods (for instancestop flow), microdialysis diffusion takes place on time periods ofthe order of minutes, the kinetic process involves the solventeffluent and the macromolecule in question and consequently itis necessary to consider the true second-order character.[5] Ac-

Wavenumber/cm−1

Tim

e/(

min

)

10

15

20

5

25

950 900 850 800 750 700

Figure 5. Changes in the 950–700 cm−1 range of poly(rC) at 3% w/wconcentration (pH 7.4) in 50 mM Tris buffer containing 0.1 M NaCl as afunction of the time of D2O efflux. Each spectrum was recorded in 1 min.

0 5 10 15 20 25

0.0

0.2

0.4

0.6

0.8

1.0

Fra

ctio

n ex

chan

ged

Time/(min)

Figure 6. Time course of deuteration of poly(rC) as measured with the784 cm−1 Raman intensity (◦) using the quartz cell and 13 kDa hollowfibre. The other symbols (•) represent the efflux of D2O and the solidline corresponds to the exchange profile calculated by Eqn (2) withkf = 1.8 min−1 and kd = 0.29 min−1.

cordingly, the biomolecular deuteration process is given by thefollowing equation:

�d(t) = 1 − kf exp(−kdt) − kd exp(−kf t)

kf − kd(2)

where �d(t) is the deuterated fraction of the biomolecularsubstance in question, and kf and kd being the biomoleculedeuteration rate and D2O efflux rate, respectively. In the case thatkf were much more greater than kd (kf � kd), the above equationwould be reduced approximately to Eqn (1), and consequently thecurves representing the solvent and biomolecular deuterationswould be very close to each other. In other words, the observedbiomolecular deuteration rate would be, in that case, limited bythe D2O efflux in this quartz cell. In order to know the limiting

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rate of biomolecular deuteration that could be resolved withthis quartz cell, a curve corresponding to the above equationwith a deuterium efflux rate of 0.29 min−1 (kd) and a second-order deuteration rate constant of 1.8 min−1 (kf ) is also includedin Fig. 6. This curve is essentially at the limit (standard errordeviation) of the experimental data and thus suggests thatthe microdialysis quartz cell of Fig. 1 is effective in monitoringexchange reactions of biomolecules with rates of 1.8 min−1

or less using 13 kDa microdialysis hollow fibres. Similarly, anexchange rate of 2.5 min−1 is the limiting rate that could beresolved with the said cell system using 18 kDa microdialysisfibres. In practice, this permits to measure proton exchangesof purine and pyrimidine bases when specific protection isafforded, for instance, by secondary structure as in the case ofZ DNA,[24,25] tertiary and quaternary structures as well as nucleicacid–protein interactions.[4] On the other hand, the exchange ratemeasured for guanine residue can vary widely as, for instance, from1.2±0.2 min−1 in some double-stranded RNAs to ∼0.16 min−1 inpoly(rG)·poly(rC).[5,26] The more rapid guanine exchange in somedouble- stranded RNAs may reflect the random distribution ofG·C base pairs within the genome RNA in question, which mayfacilitate transient base pair openings. A previous study using stop-flow UV spectroscopy showed that the exchange rate for adeninebase in poly(rA)·poly(rU) duplex was ∼1.8 min−1 at 10 ◦C.[27]

Thus, deuteration of adenine and guanine in usual Watson–Crickduplexes can also be kinetically distinguished from the solvent byRaman spectral measurements using this microdialysis quartz cell.

Conclusions

We have described a microdialysis quartz cell for use in conjunctionwith Raman spectroscopy to investigate hydrogen–isotopeexchange reactions of biomolecules, such as proteins and nucleicacids. The system requires only 40 µl volumes of the initialsubstrate and perturbing effluent solutions. The dependenceof the solvent efflux rate on the MWCO of the microdialysishollow fibre suggests to increase this parameter with the aim ofimproving the time resolution of the H/D exchange kinetics invarious structures and/or groups. We have obtained a D2O effluxrate of kd = 0.38 ± 0.008 min−1 with the greatest MWCO (18 kDa)used here, which involves that an exchange rate of 2.5 min−1

is the limiting rate that could be resolved with the said cellsystem. The deuterium efflux rates using this quartz cell are around80% of the rate values found for the capillary microdialysis cellsusing the same hollow fibre MWCO, which can be attributable tolower ratio between fibre permeable surface and sample volumeused in the quartz cell. Isotopic exchange processes with half-lives of the order of a few minutes can be resolved using the

above cell, as may occur when specific protection of purine andpyrimidine bases is afforded, for instance, by secondary, tertiaryand quaternary structures as well as by nucleic acid–proteininteractions. Although the exchange rates that can be measuredthrough this microdialysis system are relatively low, the use ofthe method described here has the advantage of avoiding sampledilution (and subsequent signal loss) involved in the known stop-flow methods.

Acknowledgement

The authors gratefully acknowledge the financial support fromthe Spanish Ministerio de Ciencia e Innovacion (Project CTQ2006-04161/BQU).

References

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