Enzymatic synthesis and characterization of L-methionine and 2-hydroxy-4-(methylthio)butanoic acid (HMB) co-oligomers

Article abstract of DOI:10.1021/jf026093g

Oligomers of L-methionine (Met) and its hydroxy analogue, 2-hydroxy-4-(methylthio)butanoic acid (D,L-HMB) were synthesized with the proteolytic enzyme papain. The Met homooligomers and HMB-Met co-oligomers obtained through the enzymatic reactions were sub

Full text of DOI:10.1021/jf026093g

J. Agric. Food Chem. 2003, 51, 2461−2467 2461
Enzymatic Synthesis and Characterization of L-Methionine and
2-Hydroxy-4-(methylthio)butanoic Acid (HMB) Co-oligomers
MATHUR RAJESH,† SHUBHEN KAPILA,*,† PAUL NAM,† DANIEL FORCINITI,†
‡
STEPHEN LORBERT,‡ AND CHARLES SCHASTEEN
Center for Environmental Science and Technology and Department of Chemistry, University of
Missouri-Rolla, 1870 Miner Circle, Rolla, Missouri 65409-0530, and Novus International, Inc., St.
Charles, Missouri 63304
Oligomers of L-methionine (Met) and its hydroxy analogue, 2-hydroxy-4-(methylthio)butanoic acid
(D,L-HMB) were synthesized with the proteolytic enzyme papain. The Met homooligomers and HMB-
Met co-oligomers obtained through the enzymatic reactions were subjected to persulfonation and
separated with reverse phase liquid chromatography (RPLC). The separated oligomers were
characterized with electrospray ionization-mass spectrometry (ESI-MS). The oligomers were also
characterized with matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-
TOF-MS). The results showed that co-oligomers were predominantly composed of 4-8 Met residues
and one HMB residue. The data also suggest that in the co-oligomers, HMB is attached at the
N-terminal end of the oligopeptide chain.
KEYWORDS: Oligopeptides; co-oligomers; methionine; methionine hydroxy analogue; MHA; HMB;
papain; mass spectrometry
INTRODUCTION
Met (Figure 1) and L-lysine are limiting amino acids in
poultry and ruminants. To enhance nutrition, these amino acids
are supplemented as crystalline amino acid salts or oligopeptides
in the diet (1, 2). However, it has been shown that the
bioavailability of orally administered amino acid salts and
oligopeptides in ruminants is very limited (1, 2). Oligopeptides
are efficiently hydrolyzed in the rumen, and the free amino acids
are readily converted to short chain fatty acids and ammonia
by rumen microbes. As a result, orally administered peptides
are not available to the animal (3). Chemical modification or
protection of the oligopeptide could render such oligopeptides
inaccessible to rumen microorganisms. A modified form of Met,
HMB (Figure 1), has been used as a Met supplement in animal
nutrition for over three decades. HMB is also referred to as
MHA in the literature. HMB itself is not an amino acid;
however, both the D and the L isomers are enzymatically
converted to Met via stereospecific amination pathways (4, 5).
Administration of HMB has been found to enhance protein
utilization, egg production, and feed conversion efficiency in
the poultry (5). HMB has been shown to be more resistant to
attack from rumen microbes than Met (6, 7). In a recent study
on HMB rumen bypass and subsequent gastrointestinal absorp-
tion of HMB, Koenig et al. (8) showed that up to 50% of HMB
escaped microbial degradation and was available for intestinal
Figure 1. Structures of Met and HMB.
absorption. However, the effectiveness of administering free
HMB has been questioned in a study on nutrition in lactating
dairy cows. The study showed that HMB supplementation did
not alter milk production or milk composition (9). To take
advantage of HMB resistance to rumen microbial attack and
enhance Met availability, administration of HMB-capped Met
oligomers has been considered. However, such oligomers are
not available; therefore, synthesis of the oligomers through
enzymatic reactions was evaluated.
The use of proteolytic enzymes for the synthesis of oligopep-
tides has been reported in a number of articles dating back to
the 1930s. It is assumed that the protease-catalyzed oligomer-
ization of hydrophobic amino acids such as Met, leucine,
tyrosine, tryptophan, and phenylalanine is driven by precipitation
of the growing hydrophobic peptide chains (10). Protease
(chymotrypsin) has been used to synthesize threonine, pheny-
* To whom correspondence should be addressed. Tel: 1(573)341-4986.
Fax: 1(573)341-6605. E-mail: kapilas@umr.edu.
† University of Missouri-Rolla.
‡ Novus International, Inc..
10.1021/jf026093g CCC: $25.00 © 2003 American Chemical Society
Published on Web 03/22/2003

2462 J. Agric. Food Chem., Vol. 51, No. 9, 2003
Rajesh et al.
lalanine, tyrosine, and Met oligomers by Brenner et al. (11, 12).
Synthesis of phenylalanine oligomers with six residues has been
reported by Dannenberg and Smith (13). Papain, a “hardy”
protease, has been widely used in the synthesis of amino acid
oligomers. Peptide synthesis with this enzyme has been evalu-
ated by Anderson and Luisi (14), who determined that the
reaction rate is dependent on the type of initiators, the ionic
strength, and the buffer composition. These parameters affect
both the yield and the polydispersity of the product (14). A
papain-catalyzed synthesis of leucine oligomers has been
described by Sluyterman and Wijdenes (15). Synthesis of
tyrosine heptamers with papain has been reported by Selvi et
al. (16). Papain and R-chymotrypsin-catalyzed synthesis of
oligo-L-glutamic acid with 5-9 residues has been reported by
Aso et al. (17). Papain has also been used by Arai et al. (18) to
catalyze synthesis of Met oligomers in a buffered medium at
pH 9; the yield of the crude oligomers was reported to be
approximately 80%. Jost and co-workers (19) synthesized Met
oligomers with the same enzyme in a 1 M citrate buffer at pH
5.5; the yield of purified oligomers was approximately 50%.
Application of papain for the synthesis of leucine, phenylalanine,
and tryptophan oligomers at pH 7.5 has also been demonstrated
by Lee et al. (20). However, enzymatic synthesis and charac-
terization of R-hydroxy acid and R-amino acid co-oligomers in
general and HMB-R-amino acids in particular have not been
described in the literature. This study was undertaken to explore
enzymatic synthesis and characterization of HMB and Met co-
oligomers.
Figure 2. Change in concentrations of monomers during the oligomer-
ization reactions with papain in a 1 Msodium citrate buffer at pH 5.5 and
37 °C. (A) Free methionine ethyl ester and Met after different incubation
periods. (B) Free HMB ethyl ester and HMB after different incubation
periods. C ) concentration of Met, methionine ethyl ester, HMB, and
t
HMB ethyl ester at time t; C ) initial concentration of free Met, methionine
0
ethyl ester, HMB, and HMB ethyl ester.
out with two procedures. In the first procedure (18), 10 g of methionine
ethyl ester or 5 g each of HMB ethyl ester and methionine ethyl ester
was dissolved in 50 mL of Nanopure water containing 0.05 mol sodium
bicarbonate buffer and 2 mmol L-cysteine. The volume of the mixture
was increased to 100 mL by adding Nanopure water. The pH of the
mixture was adjusted to 9.0, and then, 1 mL of the papain suspension
was added. After the the enzyme was added, the mixture was incubated
at 37 °C for time periods ranging from 10 min to 24 h. At the end of
the incubation period, the reaction was terminated by elevating the
mixture temperature to 80 °C for 10 min. In the second procedure (19),
3 g of methionine ethyl ester or 1.5 g each of HMB ethyl ester and
methionine ethyl ester were added to 10 mL of Nanopure water
containing 1 mmol L-cysteine, 0.1 mmol EDTA, and 0.01 mol sodium
citrate. The pH of the mixture was adjusted to 5.5 prior to the addition
of 0.5 mL of the papain suspension. The reaction mixture was incubated
at 37 °C for periods ranging between 10 min to 24 h. At the end of the
incubation period, the reaction was terminated by thermal denaturation
of enzymes at 80 °C.
Purification of Oligomers. Oligomers obtained as precipitates were
separated from the residual monomers in the supernatant through
centrifugation. The precipitate free supernatant was then filtered and
analyzed for residual esters, free Met, and HMB. The precipitate was
washed six times with 100 mL of water to remove adsorbed monomers
and salts. The precipitate was then dissolved twice in DMSO and
reprecipitated with water to remove residual HMB. The precipitate was
finally freeze-dried to obtain “purified” oligomers.
Acid Hydrolysis of Oligomers. A 25 mg aliquot of purified
oligomers was transferred to an 8 mL vial containing 2 mL of 6 N
HCl. The contents of the vial were heated to 110 °C and maintained at
this temperature for periods ranging from 24 to 168 h. To determine
the relative concentrations of Met and HMB residues in the precipitate,
aliquots of the hydrolysate were removed at different time intervals
and analyzed with the LC-DAD system.
Liquid Chromatographic Analysis of Acid Hydrolysate. An
aliquot of the acid hydrolysate was neutralized with 6 M sodium
hydroxide solution, and solvents were removed with a rotary evaporator.
The residue was dissolved in 5 mL of ethanol and filtered through a
0.2 µm membrane filter. A 10 µL portion of the filtered solution was
injected into a C-8 reverse phase column installed in the LC-DAD
system. The separation was achieved with a linear gradient program.
In the gradient, the mobile phase composition was changed from 100%
water with 0.1% TFA (eluent A) to 55% eluent A and 45% eluent B
(0.1% TFA in acetonitrile) in 30 min. The mobile phase flow rate was
Characterization of Met oligomers and HMB-Met co-
oligomers was carried out with RPLC and LC-ESI-MS. Oligo-
mers and co-oligomers were also analyzed with MALDI-TOF-
MS. These techniques are well-suited for peptide characterization,
and their application in the area is well-documented in review
articles and books (21-23).
MATERIALS AND METHODS
Materials. Met ethyl ester hydrochloride was purchased from Fluka
Chemical Corp., Milwaukee, WI. L-Cysteine, sodium citrate, TFA, and
papain (EC 3.4.22.2, twice crystallized suspension, 25 units activity/
mg, 28 mg protein/mL at pH 4.5) were purchased from Sigma Chemical
Co., St. Louis, MO. Formic acid, hydrogen peroxide, hydrobromic acid,
EDTA, methanol, and acetonitrile were obtained from Fisher Scientific,
St. Louis, MO. Phenol was obtained from Chem Service, Inc., West
Chester, PA. DMSO was obtained from Aldrich Chemical Co.,
Milwaukee, WI. Racemic HMB was procured from Novus International,
Inc., St. Louis, MO. The 250 mm × 4.6 mm i.d., 5 µm C-18 and C-8
reverse phase columns used in the RPLC separation were obtained from
P. J. Cobert Associates, Inc., St. Louis, MO.
Liquid Chromatography with DAD and MS Detection. Liquid
chromatographic separations were carried out with a model L-7000
HPLC system (Hitachi Instruments, Inc., San Jose, CA.). The system
consisted of a piston pump (L-7100), equipped with a column oven
(L-7300) and an autosampler (L-7200) with a 50 µL injection loop.
The analytes were separated on reverse phase columns and then
introduced into a DAD, model L-7450 (Hitachi Instruments). The data
were recorded with proprietary HPLC software supplied by Hitachi
Instruments. The LC-ESI-MS analysis was carried out by introducing
the column effluent into a model M-8000 ion trap MS (Hitachi
Instruments) through an ESI.
Preparation of D,L-HMB Ethyl Ester. Twenty grams of HMB was
added to 200 mL of anhydrous ethanol in a round bottom flask. The
solution was heated to 80 °C, acidified with hydrogen chloride gas
generated by adding 20 mL of sulfuric acid dropwise to 20 g of
magnesium chloride, and refluxed for 8 h, and HMB ethyl ester was
recovered by evaporating the solvent with a rotary evaporator.
Synthesis of Met and HMB-Met Oligomers. Papain-catalyzed
synthesis of Met oligomers and HMB-Met co-oligomers was carried

Methionine−Hydroxymethylthiobutanoic Acid Oligomers
J. Agric. Food Chem., Vol. 51, No. 9, 2003 2463
Figure 3. Chromatogram of free Met, HMB, and Met−Met dipeptide detected in acid hydrolysate of Met and HMB−Met oligomers. Oligomers were
synthesized with papain in a 1 M sodium citrate buffer at pH 5.5 and 37 °C. (A) Chromatogram of Met oligomer acid hydrolysate. (B) Chromatogram
of HMB−Met co-oligomer acid hydrolysate.
maintained at 1 mL/min. The eluent was introduced into the DAD and
was maintained at 1 mL/min. The eluent was introduced into the DAD
monitored over the wavelength range of 210-350 nm.
and monitored over the wavelength range of 210-350 nm.
Liquid Chromatography Mass Spectrometric Analysis of Per-
sulfonated Oligomers. To prevent the plugging problems encountered
with the use of buffered eluents in the LC-ESI-MS interfaces, the LC-
ESI-MS separations were carried out with mobile phases consisting of
water with 0.1% acetic acid (eluent A) and acetonitrile with 0.1% acetic
acid (eluent B). A linear gradient program was used to optimize
separation on a C-18 column. In the gradient, the mobile phase
composition was changed from 100% eluent A to 40% eluent A and
60% eluent B over 40 min. The mobile phase flow rate was maintained
at 1 mL/min. The LC effluent was split 4/1, and 800 µL was introduced
into a fixed wavelength UV detector set at 210 nm. The remaining
200 µL was introduced into the ESI-MS. The MS was operated in both
positive and negative ion monitoring modes over the 100-2000 amu
mass range.
MALDI-TOF-MS Analysis. Oligomer aliquots dissolved in DMSO
were analyzed with MALDI-TOF-MS using thioglycerol as the matrix.
The operating parameters of the MALDI-TOF-MS system were as
follows: acceleration potential, +20 KV; grid voltage, 80%; low mass
gate, 191.0; flight tube pressure, ∼2 × 10-7 Torr.
Persulfonation of Oligomers. Purified oligomers and co-oligomers
were persulfonated to facilitate liquid chromatographic separations.
Persulfonation was carried out in a manner similar to the one described
by Spindler et al. (24). The persulfonation involved oxidation of all
sulfide moieties to sulfones with performic acid. The performic acid
was prepared by oxidation of formic acid with hydrogen peroxide. A
0.5 mL solution of 30% hydrogen peroxide was mixed with 4.5 mL of
88% formic acid and 25 mg of phenol. The mixture was allowed to
stand for 30 min at room temperature. The mixture was then cooled to
0 °C for 15 min in an ice bath. Fifty milligrams of finely divided
oligomer powder was added to the mixture and stirred for 15 min in
the ice bath. The mixture was stored in a refrigerator overnight at 4
°C. The mixture was removed from the refrigerator, and the excess
performic acid in the mixture was reduced by adding 0.7 mL of 48%
hydrobromic acid. After the mixture was stirred for 30 min, the residual
bromine and formic acid were removed with a rotary evaporator at
50-60 °C.
RESULTS AND DISCUSSION
Synthesis of Met and HMB-Met Oligomers. The amount
of oligomer formed was determined by weighing the dried
precipitate. The extent of monomer consumption was assessed
by determining residual methionine ethyl ester, HMB ethyl ester,
Met, and HMB in the supernatant after different incubation
periods. The results of measurements for oligomer synthesized
with the procedure outlined by Jost et al. (19) are shown in
Figure 2. The results show that the concentration of methionine
ethyl ester in the reaction mixture decreased rapidly in the
presence of the enzyme. Less than 10% of the initial methionine
ethyl ester added to the mixture was found after a 6 h incubation
period (Figure 2A). The concentration of HMB ethyl ester also
decreased but to a lesser degree, and nearly 30% of the HMB
ethyl ester remained intact even after 24 h of incubation (Figure
Liquid Chromatographic Analysis of Persulfonated Oligomers.
Aliquots of persulfonated oligomers were dissolved in 5 mL of a
acetonitrile/water mixture (40:60) and filtered through a 0.2 µm
membrane filter. A 10 µL portion of the filtered solution was injected
into a C-18 reverse phase column installed in the liquid chromatographic
system. The separation was achieved with linear gradient program. In
the gradient, the mobile phase composition was changed from 100%
0.022 M phosphate buffer, pH 6.5 (eluent A), to 60% eluent A and
40% eluent B in 20 min. The eluent B consisted of 80% 0.022 M
phosphate buffer plus 20% acetonitrile. The mobile phase flow rate

2464 J. Agric. Food Chem., Vol. 51, No. 9, 2003
Rajesh et al.
Figure 4. MALDI-TOF-MS of Met oligomers and HMB−Met co-oligomers synthesized with papain in 1 M sodium citrate buffer at pH 5.5. The incubation
period was 24 h at 37 °C. (A) Mass spectra of Met oligomers. (B) Mass spectra of HMB−Met co-oligomers
2B). The results also show that the reaction was completed
within the first 6 h; after this time period, the concentrations of
free HMB and free Met remained relatively constant. These
results were in agreement with the gravimetric measurements,
which revealed that the mass of oligomer precipitates did not
increase significantly after the 6 h incubation. The results further
show that nearly 80% of Met introduced as methionine ethyl
ester is incorporated into the oligomers. The results are in
general agreement with the results obtained for Met oligomers
by Jost et al. (19) and Anderson and Luisi (14).
contained only one peak, and the retention time of this peak
was identical to that of Met. The co-oligomer hydrolysate
chromatogram contained two peaks. The retention times for the
peaks matched those of Met and HMB. The relative concentra-
tion of Met and HMB in the hydrolysate was determined to be
16:1.
MALDI-TOF-MS Analysis. The positive ion MALDI-TOF
spectrum of Met oligomers is shown in Figure 4A. The
spectrum contains ion clusters, which are 131 amu apart. This
mass difference corresponds to the repeating Met moiety (C5H9-
NOS). Because the masses of the N- and C-terminal Met
residues (C5H10NOS and C5H10NO2S) are 132 and 148,
respectively, a Met hexamer, NMet-(Met)4-MetC + H+ should
Acid Hydrolysis of Oligomers. Chromatograms of purified
Met oligomers and HMB-Met co-oligomers acid hydrolysate
obtained after 96 h of incubation are shown in Figure 3.
Chromatograms depict the output of the DAD at 210 nm. The
upper trace (A) depicts the chromatography of Met homooli-
gomers hydrolysis products, while the lower trace (B) depicts
the chromatography of HMB-Met co-oligomer hydrolysis
products. The chromatogram of Met oligomer hydrolysate
appear at m/z 805 while a heptamer, NMet-(Met)5-MetC
+
H+, should appear at m/z 936. The m/z values of the dominant
ions in the spectra did not coincide with these ions or other ion
in this series; instead, the dominant ions appeared at m/z 827,
958, 1089, 1220, 1351, and 1482. These ions most likely

Methionine−Hydroxymethylthiobutanoic Acid Oligomers
J. Agric. Food Chem., Vol. 51, No. 9, 2003 2465
Figure 5. Chromatography of oligomer sulfones synthesized with papain in 1 M sodium citrate buffer at pH 5.5 and 37 °C. (A) Met oligomer sulfones
obtained after 24 h of incubation. (B) HMB−Met co-oligomer sulfones obtained after 10 min incubation. (C) HMB−Met co-co-oligomer sulfones obtained
after 24 h of incubation.
correspond to sodiated species of the type NMet-(Met)n-MetC
+ Na+ where n ) 4-9. A second set of ions appeared at m/z
724, 855, 986, 1117, 1248, and 1379. These ions are most likely
the sodiated Met oligomer ions with an intact ethyl moiety at
the C-terminal end, NMet-(Met)n-Met-O-C2H5 + Na+. A
third set of ions appeared at m/z 740, 871, 1002, and 1135, and
these ions correspond to series NMet-(Met)n-Met-O-C2H5
+ K+. A fourth set of ions appeared at 843, 974, 1105, 1236,
and 1377, and these correspond to the series NMet-(Met)n-
MetC + K+. In all cases, the number of Met residues in the
oligomers was found to lie between 5 and 11.
A spectrum of HMB-Met co-oligomers is shown in Figure
4B. In this spectrum, ions corresponding to the series NMet-
(Met)n-MetC + Na+, NMet-(Met)n-Met-O-C2H5 + Na+, N-
Met-(Met)n-MetC + K+, and NMet-(Met)n-Met-O-C2H5
+ K+ were readily observed. However, none of the observed
ions indicated the presence of HMB residue. The apparent
absence of such ions can be attributed to the lack of a good
protonation site in HMB oligomers and/or the low concentrations
of co-oligomers of the type HMB-(Met)n in the mixture.
HPLC Separations of Met Oligomer Sulfones and HMB-
(Met)n Co-oligomer Sulfones. Chromatographic separations of
Met homooligomer sulfones are shown in Figure 5A. The
chromatogram depicts the output of the DAD at 216 nm. A
series of peaks indicating the presence of different oligomers
were observed. The chromatographic profile of the oligomers
is similar to the profile obtained by Kasai et al. (25) for Met
oligomer sulfones. The chromatographic separation of the
mixture of persulfonated Met oligomers and HMB-Met co-
oligomers is shown in Figure 5B. This chromatogram contained
a series of peaks, which were not present in the chromatogram
of the Met oligomer sulfones (Figure 5A). The extra peaks
indicate the formation of HMB oligomers or the incorporation
of HMB in the Met oligomers at the N- or C-terminal end. HMB
sulfone with a hydroxy group would be less polar than the
corresponding Met oligomer sulfone with an amine moiety.

2466 J. Agric. Food Chem., Vol. 51, No. 9, 2003
Rajesh et al.
Figure 6. Negative total ion chromatogram of oligomers synthesized with papain in 1 M sodium citrate buffer at pH 5.5 and 37 °C. (A) Met oligomer
sulfones; insert shows the mass spectrum for peak number 7, a methionine heptamer sulfone. (B) HMB−Met co-oligomer sulfones; insert shows the
mass spectrum for peak number 4′, a HMB sulfone−(Met sulfone)4.
that the degree of polymerization is dependent on the incubation
period. The intensities of longer chain oligomers were found
to increase with the incubation period. Larger oligomers were
observed in the chromatogram of oligomers obtained after 24
h of incubation as compared to the oligomers obtained after a
10 min incubation period (Figure 5B,C). Chromatographic data
also indicated that the presence of HMB might affect the relative
distribution of Met oligomers.
Table 1. LC Elution Times for Met Oligomer Sulfones and HMB−Met
Co-oligomer Sulfonesa
elution time (min)
present study
elution time (min)
Kasai et al. (25)
oligomer sulfones
(Met)4
HMB−(Met)
(Met)5
HMB−(Met)2
(Met)6
HMB−(Met)3
(Met)7
HMB−(Met)4
(Met)8
HMB−(Met)5
(Met)9
HMB−(Met)6
(Met)10
HMB−(Met)7
10.0
11.8
13.4
15.1
16.8
18.5
20.1
21.8
23.3
24.9
26.5
28.6
31.1
34.2
N/A
N/A
14.0
N/A
17.8
N/A
21.0
N/A
24.0
N/A
26.9
N/A
29.5
N/A
LC-ESI-MS Analysis of Met Oligomer Sulfones and
HMB-(Met)n Co-oligomer Sulfones. Oligomer sulfones sepa-
rated with the RPLC were characterized with ESI-MS. The ESI-
MS was operated in both the positive ion and the negative ion
mode. The HMB-containing oligomer sulfones were observed
only in the negative ion mode. The negative ion mode TICs of
the oligomers are shown in Figure 6A,B. The Met oligomer
sulfone peaks in the two sets of chromatograms could be readily
matched; the chromatogram of HMB-Met co-oligomer sul-
fones, however, contained extra peaks. The mass spectrum of
the peak with a retention time of 10.7 min in the chromatogram
of Met oligomer sulfones is shown in the Figure 6A insert.
The base ion in spectrum appeared at m/z 994, while the highest
mass ion was at m/z 1158. The difference of 164 would
correspond to the loss of Met sulfone residue from the
N-terminal end of the oligomers chain. The spectra indicate that
the peak is most likely that of a Met heptamer sulfone, (Met
sulfone)7. The spectrum for one of the “extra” peaks (retention
time of 11.8 min) in the HMB-Met co-oligomer sulfones
a N/A, not available.
Therefore, the HMB-Met co-oligomer should elute later than
the corresponding Met oligomer. The relative intensity of the
Met oligomer and possible HMB-Met peaks was found to be
3:1, respectively. Similar chromatographic results were obtained
for the HMB-Met co-oligomers synthesized at pH 9.
Chromatographic separations of persulfonated Met and
HMB-Met oligomers obtained after different incubation periods
were also performed. The results of these separations indicated

Methionine−Hydroxymethylthiobutanoic Acid Oligomers
J. Agric. Food Chem., Vol. 51, No. 9, 2003 2467
(9) Pas Bernard, J. K. Supplemental animal-marine protein blend
and a rumen protected methionine hydroxy analogue for lactating
dairy cows. Prof. Anim. Sci. 1997, 13, 149-154.
(10) Ferjancic, A.; Puigserver, A.; Gaertner, H. Papain-catalyzed
polymerization of amino acids in low water organic solvents.
Biotechnol. Lett. 1991, 13, 161-166.
chromatogram is shown in the Figure 6B insert. The base ion
in this spectrum appeared at 668, while the highest mass ion,
the likely pseudo-molecular ion, appeared at m/z 833. The
molecular ion represents persulfonated tetramethionine with one
sulfonated HMB residue, HMB sulfone-(Met sulfone)4. The
base ion at m/z 668 results from the loss of mass 165 from the
pseudo-molecular ion. This corresponds to the loss of HMB
sulfone residue from the N-terminal end of the Met oligomer
sulfones chain. The retention time of the HMB sulfone-(Met
sulfone)4 co-oligomer is higher than the Met pentamer sulfone,
(Met sulfone)5. This is in agreement with an expected increase
in retention resulting from the substitution of an amino group
with a hydroxyl group. A tentative identification of all Met
oligomer sulfones and HMB-Met co-oligomer sulfones was
made from the mass spectrometric data. These data were used
to make a tentative identification of the Met oligomer peaks
and the HMB-Met co-oligomer extra peaks in the LC-DAD
data (Table 1).
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The results indicate that the proteolytic enzyme papain can
catalyze formation of Met homooligomers and HMB-Met co-
oligomers. The total number of Met residues in the oligomers
was found to vary between 4 and 11, while only one HMB
residue was detected in each co-oligomer chain. The data also
suggest that HMB is attached at the N-terminal end of the Met
oligomer chain.
ABBREVIATIONS USED
(18) Arai, S.; Yamashita, M.; Fujimaki, M. A novel one-step process
for enzymatic incorporation of amino acids into proteins: Papain-
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Received for review November 4, 2002. Revised manuscript received
January 31, 2003. Accepted February 6, 2003.
JF026093G