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Over the past decade, there has been mounting evidencethat bacteria are a major source of marine dissolved organicmatter (Tanoue et al. 1995; McCarthy et al. 1998; Benner andKaiser 2003). D-amino acids have been identified as importantconstituents of dissolved organic matter (DOM) in the oceanand have been used to estimate the fraction of dissolved organicmatter of bacterial origin (Lee and Bada 1977; McCarthy et al.1998; Jorgensen et al. 1999; Dittmar et al. 2001).

Bacterial membranes are considered the main source ofD-amino acids (Salton 1994). As components of the inter-peptide bridge in peptidoglycan, they crosslink the glycanstrands of N-acetyl-glucosamine and N-acetyl-muramic acidand so contribute to the overall rigidity of the cell mem-brane. Because the chiral amino acids are linked throughamide and glycosidic bonds, strong acid hydrolysis isrequired to release individual enantiomers (Schleifer and Kan-dler 1972; Labischinski and Maidhof 1994).

An inherent problem of acid hydrolysis is that it inducesoptical inversion of amino acids. Thus, the measured concen-tration of the individual enantiomer always includes a frac-tion derived from racemization of the other enantiomer. Tocomplicate matters, the rate at which amino acids isomerize

during acid hydrolysis depends on the molecular structure ofthe amino acid polymer, the position of the amino acid in thepolymer and the sample matrix (Mitterer 1972; Kvenvolden etal. 1973; Schroeder and Bada 1976; Smith and De Sol 1980;Liardon et al. 1981; Bada 1984). Intramolecular reactions, fieldand resonance effects, inductive factors, and steric hindranceinfluence the energy of the racemization transition stateeither enhancing or slowing racemization (Smith and De Sol1980; Smith and Reddy 1989).

A variety of techniques using hydrochloric acid hydrolysiswith liquid chromatography (Bruckner et al. 1991; Mopperand Furton 1991; Fitznar et al. 1999), gas chromatography(Hoopes et al. 1978), and electrophoresis (Wan and Blomberg2000) do not account for acid-induced racemization. As aresult, reported values for D- and L-enantiomers of amino acidsdo not include a correction for the hydrolysis effect and couldlead to quantification errors. In order to correctly determineacid-induced racemization, several analytical approachesincorporate a hydrolysis in deuterated acid to identify theenantiomers produced by racemization (Frank et al. 1979; Liar-don et al. 1981; Goodlett et al. 1995). Amelung and Brodowski(2002) report problems using a single hydrolysis with deuter-ated HCl and propose a dual hydrolysis, one with HCl and oneparallel with deuterated HCl. Their method is not limited byside chain incorporations of the deuterium label and so can beapplied to acidic amino acids (Amelung and Brodowski 2002).

A different concept involves a time series of hydrolyses for thesame sample and linear extrapolation to time 0 h to quantify theinitial concentration of D-enantiomers (Nagata et al. 1998).

Hydrolysis-induced racemization of amino acidsKarl Kaiser* and Ronald BennerDepartment of Biological Sciences, University of South Carolina, Columbia, SC 29205, USA

AbstractAcid hydrolysis–induced racemization compromises accurate determination of enantiomeric amino acid

compositions. In the present study, the extent of amino acid racemization during a vapor (6 M HCl, 32.5 min,150°C) and liquid-phase hydrolysis (6 M HCl, 20 h, 110°C) was assessed, and the influence on the accuracy ofenantiomer measurements was investigated. The extent of hydrolysis-induced racemization was controlled bythe molecular structure of the amino acid polymer and hydrolysis conditions. Vapor-phase hydrolysis showedsignificantly higher racemization compared to liquid-phase hydrolysis. Without correction, hydrolysis-inducedracemization resulted in overestimation of D-enantiomers and could lead to erroneous conclusions about theoccurrence of D-enantiomers in natural samples. The range of racemization was determined with proteins andfree amino acids, and an averaged racemization value is proposed to account for enantiomer exchange duringhydrolysis. In addition, a microwave-assisted vapor-phase hydrolysis is described that allows preparation of 40samples in about 3 h with very low blanks.

*E-mail: karl@biol.sc.edu

AcknowledgmentThis research was supported by National Science Foundation Grant

OCE 0080782.

Limnol. Oceanogr.: Methods 3, 2005, 318–325© 2005, by the American Society of Limnology and Oceanography, Inc.

LIMNOLOGYand

OCEANOGRAPHY: METHODS

However, this method relies on a linear behavior of amino acidracemization over the time of the hydrolysis. Nonzero interceptssuggesting the presence of D-amino acids could also be causedby initial low cleavage of peptide bounds or a changing racem-ization mechanism over time (Frank et al. 1981).

In this study, we describe a critical assessment of acidhydrolysis induced racemization and the effect on the deter-mination of the original amino acid enantiomer composition.A mathematical formula is derived to correct analyzed samplesfor the estimated fraction of enantiomers that are produced bythe hydrolysis. Additionally, a microwave-assisted vapor-phasehydrolysis that facilitates rapid and simple sample preparationwith exceptionally low blanks is described.

Materials and proceduresChemicals—o-Phthaldialdehyde (OPA), N-isobutyryl-L-cys-

teine (IBLC), N-isobutyryl-D-cysteine (IBDC), D- and L-aminoacid kits, L-glutamic acid-methylester, lysozyme, bovineserum albumine, and Bacillus subtilis (ATCC 13430, type II)were obtained from Sigma. Hydrochloric acid A.C.S plus(36%), potassium di-hydrogen phosphate, boric acid, 50 w/w% NaOH, and methanol were purchased from Baker. Ampules(2 mL), micro-pipettes (50 µL), and nitrile gloves were fromFisher. Auto sampler vials (2 mL, 12 × 32 mm), flat bottomglass micro inserts (400 µL), and screw caps with Teflon-linedsilicone septa were purchased from Agilent. Synechococcusbacillaris was grown in an artifical seawater medium (Bid-danda and Benner 1997). Axenically grown phototrophicalgae, Emiliania huxleyi (CCMP 373), Thalassiosira oceanica(CCMP 1005), Nannochloris sp. (CCMP 518), and Heterocapsaeniei (CCMP 447), were obtained from Bigelow. Seawater sam-ples were from Station Aloha, Hawaii.

OPA/IBL(D)C reagents were prepared fresh every 3 d. Into100 µL methanol, 5 mg mL–1 IBL(D)C and 2 mg mL–1 OPA weredissolved and diluted with 900 µL 0.5 M boric acid buffer(pH = 9.5). The reagent was stored at 4°C.

Microwave-assisted vapor-phase hydrolysis—Dissolved aminoacids were hydrolyzed in a CEM Mars 5000 microwaveequipped with a protein hydrolysis accessory kit (Jorgensenand Jensen 1997). The protein hydrolysis kit included fourTeflon vessels for the samples and one Teflon vessel for thetemperature sensor, pressure sensor, and a connection to anitrogen tank. Custom-made Teflon inserts accommodated 10flat bottom glass micro inserts (400 µL) per vessel, and 40samples could be hydrolyzed at the same time. The hydrolysisprocedure allowed for very low blanks if properly conducted.Blanks, including amino acids from reagents, usually werebetween 10 and 20 nM. Several steps were crucial for these lowblanks. Before hydrolyzing samples, a blank hydrolysis wasperformed. This was also helpful to test for leaks in the vesselsetup. Most important was that all equipment touching themicro inserts was clean, either acid washed and methanolrinsed or muffled. Micro-capillaries (50 µL), which directedthe stream of nitrogen during the drying of samples, were

muffled before use and frequently exchanged. After the dryingstep, the micro inserts were transferred with clean forceps intothe hydrolysis vessel. All sample handling was done in a lam-inar flood hood.

Hydrolysis conditions were optimized with bovine serum albu-min and surface and deep seawater. Before hydrolysis, 12 mMascorbic acid (10 µL mL–1) was added to samples to preventoxidation of amino acids by nitrate (Robertson et al. 1987).Typically, 100 µL samples were pipetted into 400-µL flat bot-tom micro inserts and dried with a gentle stream of nitrogen.Flat bottom micro inserts were used to allow for maximalexposure of the dried sample to acid vapor. Hydrolysis vesselswere filled with 5 mL of 6 M hydrochloric acid and the microinserts were added with a clean forceps into the custom-madeholder. The vessel tops were tightened with a CEM cappingstation to ensure a constant torque and to prevent leaks. Afterarranging the vessels in a Teflon holder, the setup was placedin the microwave and the pressure and nitrogen line were con-nected. The thermo-sensor was fit into a glass tube that was incontact with the acid in the vessel. Before the hydrolysis wasstarted, all the vessels were evacuated followed by flushingwith nitrogen at 15 psi. After five repeats of evacuation andflushing, pressure was kept at 15 psi. The following hydrolysisconditions were used: Microwave power output = 600 W, rampto 150°C in 3 min, hold for 32.5 min at 150°C, cool down to90°C before terminating the run. Before opening, the vesselswere carefully leaned at a 45° angle and gently tapped on thetable surface to remove any condensed drops of acid on thelid. Acid moisture was first blown off with nitrogen, then 20 µLof water was added, and samples were redried.

Liquid hydrolysis—Microwave-assisted vapor-phase hydrolysisfailed to efficiently release amino acids from particles. There-fore, the traditional liquid-phase hydrolysis in 6 M HCl wasemployed for solid samples (Henrichs 1991). Particulate sam-ples were weighed into ampules and 500 µL 6 M HCl and 5 µL12 mM ascorbic acid were added. The ampules were flushedwith nitrogen, sealed, and placed in an oven at 110°C. After 20h, the hydrolysis was terminated in a cold water bath. Thehydrolysate was usually diluted 3 times with water, centrifugedto remove all particles, and 20 µL were dried with a stream ofnitrogen. To ensure removal of HCl, 20 µL of water were addedtwice and the sample was redried after each addition.

Chromatography—Enantiomeric amino acids were sepa-rated on a Licrospher 100 RP18 (250 × 4 mm, 5 µm) with aguard column (5 × 4 mm) after in-line derivatization withOPA/IBLC or OPA/IBDC at 20°C. The system was an AgilentHP 1100 system with an auto sampler and fluorescencedetector controlled by HP Chemstation software. Afterhydrolysis, dry and neutral samples were dissolved in 100 µLwater and 5 µL of L-glutamic acid-methyl-ester, the internalstandard, was added. Vigorous vortexing usually dissolvedthe entire dried residue in the micro-inserts. Insoluble parti-cles were removed by centrifugation and the sample wascarefully transferred to a new micro insert if necessary. To

Kaiser and Benner Hydrolysis-induced amino acid racemization

319

facilitate separation of enantiomeric amino acids, a secondchiral center was introduced by derivatization with IBLC andIBDC. Samples were run with both reagents, which allowedfor correction of co-eluting peaks.

A total of 30 µL of sample and 20 µL of IBL(D)C reagentwere drawn into the sample loop and mixed inline for 2 min.After injection, the gradient program was started. For sampleswith > 500 nM individual amino acids, 10 µL of sample weremixed with 10 µL of 0.5 M borate acid buffer (pH = 9.5), and10 µL IBL(D)C reagent. Samples were run alternating first withOPA/IBLC then OPA/IBDC.

Method program and gradient: Solvent A was 40 mM potas-sium di-hydrogen phosphate adjusted to pH 12. Solvent B wasmethanol:acetonitrile (13:1 v/v). Amino acid derivatives wereseparated with a linear binary gradient starting with 100% Ato 61% A at 50 min, then 46% A at 72 min, and 40% A at 80min. After 80 min, the system was returned to 100% A andequilibrated for 3 min. The flow rate was 0.8 mL min–1, andtotal run time was 87 min. Excitation was at 350 nm andemission of OPA derivatives was recorded at 420 nm.

Quantification—Varying concentrations of standards (n = 3)spiked with the internal standard (ISTD) were measured andrelative response factors for each amino acid (RRFx) were cal-culated (excluding the origin).

. (1)

Individual amino acid concentrations in samples were thendetermined according to

. (2)

The procedural blank (Milli-Q Plus UV water) was analyzedalong with samples and subtracted from concentrations ofamino acids calculated using Eq. 2.

AssessmentMicrowave-assisted vapor-phase hydrolysis—Vapor-phase

hydrolysis combined with microwave heating provided a rela-tively fast and simple tool to hydrolyze amino acids. Optimalhydrolysis conditions were identified with surface and deepocean water from the North Pacific. The efficiency of deter-mined conditions was tested with dissolved bovine serumalbumin. Triplicate samples were hydrolyzed and the averageamino acid recovery was 101% ± 3% based on the knownstructure of this protein (Reeck 1976). Vapor-phase hydrolysisof particulate samples generally recovered lower amounts ofamino acids compared to liquid-phase hydrolysis limiting theuse of vapor-phase hydrolysis to dissolved samples (data notshown). We did not compare vapor-phase and liquid-phasehydrolyses for seawater DOM, but it has been reported thatvapor-phase hydrolysis yielded up to 300% higher amino acidconcentrations in seawater compared to traditional liquidphase hydrolysis (Keil and Kirchman 1991).

Blank considerations—Total hydrolyzable amino acid concen-trations in deep ocean water from the North Pacific were 57± 4 nM (n = 4). At such low concentrations, careful determina-tion of the procedural blank was important for the accuracy andprecision of the method. Procedural blanks were run parallelwith samples and resulted from sample handling, hydrolysis,and chemicals that were used for derivatization and buffers. Itwas noticed that the IBL(D)C reagents contained measurableconcentrations of racemic alanine. The procedural blank waslower with the IBLC reagent compared to the IBDC reagentbecause of less D- and L-alanine and typically ranged from 10 to20 nM. Lowest blanks were achieved with fresh reagents andthoroughly cleaned equipment. Working in a laminar flowhood considerably improved the precision of the method.

Chromatography—Chromatographic separation of enan-tiomeric amino acids was achieved by derivatization with achiral thiol/OPA reagent resulting in diastereomeric aminoacids that separated on a reversed phase column (Bruckner etal. 1991; Fitznar et al. 1999). Samples were run with the L- andD-enantiomer of the reagent to identify co-elution problems.Switching the reagent reversed the retention times of thediastereomeric amino acids pairs. It was assumed that onlychiral amino acids were sensitive to this change and not co-eluting achiral compounds. If comparison of the two runsidentified a co-elution problem, the lower value was recordedand used for racemization correction.

Precision and limit of detection—The precision of vapor-phasehydrolysis and chromatographic analysis was evaluated withreplicates of seawater from the North Pacific (n = 3). At 50 to100 nM concentrations, the relative standard deviation was5% to 12%, and at 5 to 10 nM concentrations, the relativestandard deviation was 11% to 25% for individual aminoacids. Precision values included day-to-day variabilitybecause replicates were run on different days. Limits of detec-tion (S/N = 3) were 4 to 29 fmol for individual amino acids.Liquid-phase hydrolysis with chromatographic analysisshowed similar precision intervals and limits of detection.

Characteristics of hydrolysis-induced racemization—Acid-induced racemization was determined for microwave-assistedvapor-phase hydrolysis and liquid-phase hydrolysis usingmonomeric amino acids and two different proteins. Table 1shows percentages of individual D-enantiomers produced dur-ing microwave-assisted vapor-phase hydrolysis and liquid-phase hydrolysis of L-enantiomers. It was assumed that bothproteins originally did not contain any D-forms of aminoacids. As acid-catalyzed deamidation transformed asparagineand glutamine into the corresponding acids, concentrationsof aspartic acid and glutamic acid included both amino acids.Not included were threonine and isoleucine because racem-ization produced the diastereomer, allo-threonine, and allo-isoleucine, respectively, which were not completely resolvedin the chromatogram. Methionine and tryptophan were omit-ted because both hydrolyses were inefficient in recoveringthese amino acids.

Amount of AA(x)Amount of ISTD Response

Respons= × x

ee RRFISTD x×

RRFResponse Amount

Response Amounxx ISTD

ISTD

= ×× tt x

Kaiser and Benner Hydrolysis-induced amino acid racemization

320

Important results of this comparison were that (1) racemiza-tion of amino acids was substantially higher with microwave-assisted vapor-phase hydrolysis compared to liquid-phasehydrolysis. Microwave heating at higher temperatures acceler-ated racemization of amino acids leading to conversion of indi-vidual L-enantiomers as high as 10%. (2) In both hydrolyses,racemization was significantly different between proteins andfree amino acids indicating the influence of the molecular struc-ture on racemization. (3) Trends for individual amino acids weresimilar in both hydrolyses. Aspartic acid and glutamic acidshowed the highest racemization induced by the second car-boxyl group that stabilized the transient enolate (Frank et al.1981). Serine and valine were highly resistant to racemization.

As expected, racemization values in axenic phototrophicalgae were within the range of free amino acids and protein-bound amino acids (Table 1). Amino acids in phototrophicalgae are usually bound in proteins with a small cellular frac-tion existing in the free form. Racemization values in thesealgae, therefore, represented an average of all possible struc-tures. Published racemization values derived from a variety ofproteins with similar liquid-phase hydrolysis conditions werein good agreement with values in Table 1 (Manning 1970;Frank et al. 1981; Liardon et al. 1981; Csapo and Csapo-Kiss1998). This indicated that Table 1 presented a maximal rangeof racemization induced by hydrolysis conditions.

All racemization estimates were based on inversion ofL-enantiomers to D-enantiomers because no proteins wereavailable containing exclusively D-enantiomers. Since themain sources for D-amino acids are cell wall peptides (Salton1994), racemization is most likely similar to proteins. No differ-ence was observed between racemization of free L- or D-aminoacid enantiomers (data not shown).

Racemization correction—Stereochemical inversion of aminoacids can be described by first-order kinetics (Liardon et al. 1981):

Therefore, the integrated expression of the racemization ofenantiomers, with the first- order rate constant k = k1 = k2, is

. (3)

As samples contain D- and L-enantiomers, measured enan-tiomeric amino acid concentrations are always composedof original enantiomers and enantiomers resulting fromhydrolysis-induced racemization. For the L-enantiomerthis means

(4)

where L* and D* are the measured concentrations, and L*0 and D*

0

are the original concentrations. The sum of measured amountsof both enantiomers is equal to the sum of original enantiomersassuming no enantiomers are destroyed during hydrolysis.

(5)

To calculate original enantiomer concentrations, D*0 in Eq.

4 is substituted with Eq. 5. For the L-enantiomer, this leads to

(6)

Term e-2kt describes the extent of racemization for individ-ual amino acid enantiomers dependent on hydrolyses condi-

LL e D e

e

kt kt

kt0

2 2

2

1 1

2*

* *

=+( ) − −( )− −

−

D L D L0 0* * * *+ = +

L Le

Dekt kt

* * *= +

+ − +

− −

0

2

0

21

21

1

2

eD L

D Lkt− = −

+2 1

1

L enantiomer D enantiomerk

k

− ↔ −2

1

Kaiser and Benner Hydrolysis-induced amino acid racemization

321

Table 1. Percentages of D-enantiomers (%D) produced during acid hydrolysis of L-enantiomers

Vapor-phase hydrolysis Liquid-phase hydrolysisFree Free Phototrophic AA* BSA* Lysozyme* Average† AA* BSA* Lysozyme* Average† algae‡

Asp 8.5 (0.3) 10.3 (0.6) 8.8 (0.8) 9.4 (0.4) 3.2 (0.0) 5.1 (0.1) 5.6 (0.2) 4.4 (0.1) 3.0 (0.4)

Glu 4.6 (0.1) 7.2 (0.1) 4.3 (0.6) 5.9 (0.1) 1.4 (0.2) 2.6 (0.1) 2.5 (0.0) 2.0 (0.1) 2.5 (0.1)

Ser 1.1 (0.2) 1.5 (0.2) 0.9 (0.1) 1.3 (0.2) 0.0 (0.0) 0.0 (0.0) 0.6 (0.0) 0.3 (0.0) 0.4 (0.1)

His 4.8 (0.3) 7.1 (0.1) 0.0 (0.0) 6.0 (0.2) 2.6 (0.0) 3.8 (0.0) 2.7 (0.0) 3.2 (0.0) ND

Arg 3.6 (0.1) 5.4 (1.1) 6.4 (1.0) 5.0 (0.5) 1.0 (0.0) 2.0 (0.2) 2.8 (0.4) 1.9 (0.2) 3.2 (0.6)

Ala 2.7 (0.1) 5.8 (0.0) 3.8 (0.5) 4.3 (0.1) 0.6 (0.0) 1.9 (0.0) 1.7 (0.1) 1.2 (0.0) 1.8 (0.1)

Tyr 4.3 (0.1) 4.2 (1.5) 4.1 (0.9) 4.2 (0.8) 2.1 (0.0) 1.2 (0.0) 0.0 (0.0) 1.7 (0.0) 2.1 (0.3)

Val 0.7 (0.1) 2.4 (0.0) 1.3 (0.3) 1.6 (0.0) 0.0 (0.0) 0.0 (0.0) 2.8 (0.2) 1.4 (0.1) 2.8 (0.7)

Phe 2.5 (0.1) 8.1 (1.2) 5.1 (0.1) 5.3 (0.7) 1.0 (0.0) 2.5 (0.5) 0.6 (0.0) 1.7 (0.3) 0.4 (0.1)

Leu 2.8 (0.1) 7.4 (0.9) 5.4 (1.1) 5.1 (0.5) 0.9 (0.0) 2.1 (0.0) 2.2 (0.1) 1.6 (0.1) 2.0 (0.1)

Lys 2.2 (0.4) 4.1 (0.0) 2.5 (0.6) 3.2 (0.2) 1.3 (0.0) 1.5 (0.1) 2.1 (0.2) 1.7 (0.1) ND

*Values are calculated as %D = 100*D/(D + L); mean errors (n = 2) are in parentheses.†Average calculated from highest and lowest racemization of the proteins (BSA, lysozyme) and free amino acids; mean errors in parentheses include meanerrors of highest and lowest racemization values.‡Phototrophic algae values are averages from the analysis of 4 axenic cultures (Emiliania huxleyi [CCMP 373], Thalassiosira oceanica [CCMP 1005], Nan-nochloris sp. [CCMP 518], and Heterocapsae niei [CCMP 447]) with standard errors in parentheses. ND = not determined.

tions, molecular structure, and matrix composition. Eq. 6combined with Eq. 3 gives

. (7)

The D/L ratio was estimated with free amino acids and twodifferent proteins (Table 1, D/L = %D/(100 – %D)). OriginalD-enantiomer concentrations were calculated by rearrangingEq. 5:

. (8)

A simple model was used to examine the influence ofracemization on measured amino acid enantiomers. D- andL-enantiomer concentrations of aspartic acid, glutamic acid,serine, and alanine were varied in small increments startingfrom a mixture containing equal amounts of both enan-tiomers to decreasing amounts of the D-enantiomer. The mix-tures were corrected for hydrolysis-induced racemization withan averaged racemization estimate calculated from free aminoacid and protein racemization values summarized in Table 1.As can be seen in Fig. 1, the relative difference between cor-rected and uncorrected values for a mixture dramaticallyincreases at disparate enantiomer concentrations. This trend ismagnified for vapor phase hydrolysis due to enhanced racem-ization of amino acids (Fig. 1B). Results clearly demonstratethat hydrolysis-induced racemization leads to large errors atlow percentages of one enantiomer if racemization correctionis omitted. Differences between individual amino acids reflectdissimilar racemization characteristics.

D-amino acids in natural samples—To explore the effect ofacid-induced racemization on measured D-amino acid concen-trations in natural samples, two different bacteria, B. subtilis(Gram positive) and S. bacillaris (Gram negative), and seawaterfrom the North Pacific were hydrolyzed in liquid or vapor-phase. Gram-negative bacteria are the dominant bacteria in sea-water and marine sediments (Moriarty and Hayward 1982;Giovannoni and Rappe 2000), and bacterial cell walls are con-sidered major sources for D-amino acids (Salton 1994). OriginalD-amino acid enantiomers were calculated using Eqs. 7 and 8with racemization estimates derived from free amino acids andproteins. The amino acid composition in S. bacillaris, a marineGram-negative phototrophic bacterium, was dominated by L-amino acids. Acid-catalyzed racemization contributed a majorfraction of total measured D-amino acids (Fig. 2A). Racemiza-tion could account for all D-enantiomers of aspartic acid, serine,valine, phenylalanine, and leucine if the highest possibleracemization was assumed. Only glutamic acid, arginine, andalanine gave D-enantiomer concentrations that were differentfrom zero indicating that the sample contained native D-enan-tiomers. However, minimal estimates were 3- to 7-fold lowerthan maximum estimates, and accurate quantificationremained elusive. Values for histidine, tyrosine, and lysine werenegative after racemization correction, which was interpreted ascomplete absence of D-enantiomers. Similar results wereobserved in a different Gram-negative phototrophic bacterium,Trichodesmium sp., clearly demonstrating the inability of thismethod to accurately quantify enantiomeric amino acids if oneenantiomeric form dominated the composition.

In contrast to S. bacillaris, the Gram-positive soil bacteriumB. subtilis showed relatively high percentages of D-enantiomersfor alanine and glutamic acid (Fig. 2B).

Ranges introduced by different racemization correctionswere 27% to 28% and 15% to 16% D-enantiomers, respec-

D D L L0 0* * * *= + −

LL D D L

D L0 1*

* * ( )= −−

Kaiser and Benner Hydrolysis-induced amino acid racemization

322

Fig. 1. Difference between measured percentages and racemization cor-rected percentages of D-enantiomers for (A) liquid phase hydrolysis and(B) vapor phase hydrolysis. Average racemization estimates from Table 1were used for correction: %D = 100*D/(D + L).

tively. Results also indicated small amounts of D-aspartic acid,serine, and arginine. However, D-aspartic acid values variedfrom 1% to 4% with most of the D-enantiomers possiblyoriginating from racemization. Similar trends were observedfor D-serine and D-arginine. Percentage ranges of D-histidine,D-tyrosine, D-valine, D-phenylalanine, D-leucine, and D-lysinesuggested that these D-isomers could be solely derived fromhydrolysis effects.

A surface seawater sample from the open ocean was chosento identify the effect of microwave-assisted vapor-phase hydrol-ysis on D-enantiomer concentrations of individual amino acids(Fig. 2C). This sample contained relatively high percentages of

D-aspartic acid, glutamic acid, serine, and alanine. Includingreverse racemization, the range for these D-enantiomers waswithin 1% to 12% of the average and similar to the analyticalerror. Other D-amino acids could not be precisely quantifiedbecause concentrations were between the limit of detection(S/N = 3) and the limit of quantification (S/N = 10).

DiscussionMicrowave-assisted vapor-phase hydrolysis—The major advan-

tages of microwave vapor-phase hydrolysis are (1) it allows pro-cessing of 40 samples in about 3 h, (2) the volume of sampleneeded for analysis is typically 100 µL, (3) samples after hydrol-ysis contain only hydrochloric acid moisture, and (4) blanksthat include amino acids from reagents and solvents are as lowas 10 nM total amino acid concentration. Accordingly, thishydrolysis is well suited for routine analysis of dissolved sam-ples and especially valuable if only small amounts of sampleare available.

Racemization correction of measured enantiomer concentrations—Hydrolysis-induced racemization is an inherent problem ofanalytical techniques used for measuring amino acid iso-mers. The correction for this racemization effect ultimatelydetermines the accuracy of individual enantiomer mea-surements. Hydrolysis tests with proteins and free amino acidsdemonstrated the wide range of stereochemical inversion ofamino acids indicating production of substantial amounts ofD-enantiomers from the corresponding L-enantiomers duringacid hydrolysis. The degree of racemization was controlled byhydrolysis conditions and the molecular structure of theamino acid polymer. Racemization was higher if a vapor-phasehydrolysis combined with microwave heating was used. Inprotein structures, up to 10% of L-aspartic acid enantiomerswere inverted by this acid hydrolysis. Liquid-phase hydrolysisinduced less racemization but still led to considerableamounts of converted enantiomers (Table 1).

The exact molecular structures and matrix compositions ofmost samples are unknown so accurate assessment of hydrol-ysis-induced racemization is only possible if tedious isotopiclabeling techniques are used. Hydrolysis-induced racemizationimpacts accurate determination of amino acid enantiomersespecially at disparate concentrations of individual enan-tiomers (Figs. 1 and 2). One approach for racemization correc-tion of amino acid enantiomer measurements in natural sam-ples is to establish a maximal range of hydrolysis-inducedracemization with free amino acids and proteins and then usethe average of the two extremes to correct measured enan-tiomer concentrations.

Intramolecular effects in intact protein structures are impor-tant for stabilizing the racemization transition state and, there-fore, enhance racemization (Smith and De Sol 1980; Smith andReddy 1989). A major fraction of marine dissolved organicmatter and amino acids consist of < 10 monomeric subunits(Benner et al. 1997; Kaiser and Benner unpubl. data unref.),suggesting racemization rates are most likely lower than maxi-

Kaiser and Benner Hydrolysis-induced amino acid racemization

323

Fig. 2. Amino acid D-enantiomer compositions (%D) of (A) S. bacillaris,(B) B. subtilis, and (C) open ocean surface water from the North Pacificbefore and after racemization correction. S. bacillaris (A) and B. subtilis (B)were hydrolyzed in liquid phase (6 M HCl, 20 h, 110°C). Open ocean sur-face water was hydrolyzed with a vapor phase method (6 M HCl, 32.5min, 150°C).

mal racemization values found in proteins. Marine particulateand dissolved organic matter share compositional similaritiesthat are rapidly imprinted by decomposition processes (Wake-ham et al. 1997; Meon and Kirchman 2001). Soil organic mat-ter comprises a variety of molecular compounds ranging fromphotosynthate to highly degraded components (Hedges andOades 1997) with overall racemization rates potentially lowerthan unaltered proteins. Therefore, most environmental sam-ples very likely display racemization characteristics somewherebetween intact proteins and free amino acids.

In this study, the proteins lysozyme and bovine serum albu-min were used to estimate the greatest extent of hydrolysis-induced racemization. To evaluate whether racemization esti-mates derived with these proteins and free amino acids describea maximal range of acid-catalyzed inversion, racemization esti-mates were compared to racemization in axenic phototrophicalgae and literature values. Published hydrolysis-induced racem-ization of amino acids in a variety of proteins overlapped withracemization values presented in this study (Manning 1970;Frank et al. 1981; Liardon et al. 1981; Csapo and Csapo-Kiss1998). Axenic phototrophic algae, comprised of a diverse mix-ture of different proteins and a small fraction of free amino acids,yielded racemization values very similar to those from analyzedproteins. These results suggest the average hydrolysis-inducedracemization values and Eqs. 7 and 8 presented in this study areapplicable to other studies using similar hydrolysis conditions.

ReferencesAmelung, W., and S. Brodowski. 2002. In vitro quantification

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Submitted 24 February 2005

Revised 14 June 2005

Accepted 13 July 2005