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Total hydrolysis of proteins with strongly reduced racemization of amino acids
200
Btochmaca el Btophvswa Acta, 1037 (19qO) 200-208 El.~evaer
B~APRO 33544
Total hydrolysis of proteins with strongly reduced racemization of amino acids Antimo D'Aniello, Guiseppe D'Onofrio and Michele Pischetola Department of fliochemlst~,, Sla~lone Zoologwa "A Do~rn : Vdla Comunale, Naples (Italy') (R~et~ed 27 June 1999)
Key gords: Attune acids racemw.ation: D-Aspartate: D-Amino acid: Protein hydrolysis; D-Amino aod hydrolysis; (Human cata:~l lens)
A new method has been devised for the complete hydrolysis oi pcoteins with an extremely low level of racem/z'z~i~a ol amino acids. Proteins are incubated in 10 M HCI at • low temperature to obtain partial hydl~lysis, qIhey are then incubated with pconase and finally with leucine a m i ~ and peptidybD-amino-acid hydrolnse from vu/~m'& The proposed method ensures the total hydrolysis of either puriCM~dpcoteins or protei~ contained in • crude homogenate of animal or vegetable tissue. In both cases, the racemization of ,.alao acids (expressed as rate d D f o r m / o + L form X 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. D-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus D-aspartate oxidase or hog kidney D-amino acid oxidase; with tlbe~ e n z y m ~ 0.05 nmoi o~ • D-am/tao acid was d~ecmimsd in the presence of up to 20 /~mol of a mixture ol L-amino acids (ratio % D / D + L =0.00025). The met4md allows ~ e determination of l>-amino acids either in tissues in which they are present in high ctmcentratioas (as human cataract lenses, tooth enamel, etc.) or in those with low emmtlomer content (as brain erythrocytes, etc.). Using the described, we hydrolyzed several synthetic peptides consisting el i>. and L-amino acids and d e t e n n i n ~ the a m m ~ d D-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human eataractous lenses. The amount og D-aspartic acid was 0.026 lamoi/mg in lenses of women aged between 71 and 76 years and 0.07~6/zmol/mg in lenses of men aged between 55 and 72 years. The D-aspartlc acid measured corresponds to about 12% with respect to total aspartie acid, Introduction The purpose of the present work was to determine the content o1 D-amino acids in pzptides and proteins to a high level of accuracy. A preliminary step of ',his analysis is to completely hydrolyzc these compounds to obtain free amine adds from which the D-amino acids levels can then he determined. The chemical metho.'i normally used to hydrolyze proteins (6 M HCI at ~10 °C for 24 h) provokes racemization of amino acids [1-4]; and enzymatic hydrolysis, which does not cause racemization of amino acids, does not bring about total hydrolysis of proteins. As far as we are aware, apart from the complete hydrolysis of cytochrome c [5], there is no other example of complete hydrolysis of proteins using only hydrolases. In ad-
Correspondence: A. D'Anidlo, Stazion¢ Zo~loglca "A. Dohm', Villa Comunale, 80121 Napoli, Italy.
difon, no one hydrolytic enzyme yet studied is able to cut peptide bonds consisting of D-amino acids. In a previous study, we purified peptidyi-l>-aminoacid hydrolase from the intestine of the Loligo t,ulgaris that hydrolyzes small peptides consisting of D- or Lamino acids [6]. This prompted us to study whether rids enzyme could be used to free D-amino acids from their proteic compounds. In this work we describe a new method for t~he complete hydrolysis of proteins either in a purified form or contained in whole animal and vegetable tissues which concomitantly strongly reduces racemization of amino acids. This method is carried out firstly by chemical hydrolysis in I0 M HCI at a low temperature to obtain a small proteic fragment and then continuing hydrolysis using, pronase. Finally, leucine aminopeptida~ with peptidyl-l>-amino-acid hydrolase complete the hydrolysis. Using this procedure we were able to determine precisely the D-amino acid content in various peptides and proteins. These results induced us to conclude that
016%4838/90/,933.50 ~: 1990 Elsm'ier Science Pubhshers B.V. ~,Biomcdical Dies,on)
201 the present method may be considered as a general procedure to determine D-amino acids in proteic co,mpounds.
Materials and Methods Chemicals Peroxidase from horseradish (250 U/mg. 10 mg/mi, suspension), malate dehydrogenase (1200 U / m g . 10 mg/ml, solution in glycerol, 50%), lactate dehydrogenase (550 U / m g , 10 mg/ml, solution in glycerol, 50%), papain (EC 3A.22.2), chymotrypsin A4 (EC 3.4.21.1), tryosin (EC 3.4.21.4). neutral proteinase (EC 3.4.24.4), pronase from S. griseus, carboxypeptidase (EC 3.4.12.1), D-amino-acid oxidase (EC 1.4.3.3) from hog kidney, and reduced ~-nicotinamideadenine dinucleotide (/]-NADH) disodium salt. were purchased from Boehringen Mannheim. Uhrafihration membrane cones type CF 25 were from Amicon, U.S.A. Leucine aminopeptidase (EC 3.4.1 !.1 ). peroxidase (EC 1.11.1.7) from horseradish, 10 m g / m i (2500 U/ml). catalase (EC 1.11.1.6) from bovine liver 15 mg/ml (50000 U/rag) and all other chemical used in this work were from Sigma. St. Louis. MO, U.S.A. En:yme preparations Pronase (approx. 7000 U / g tyophilisate). In order to eliminate possible contamination by free amino acids or peptides contained in the lyophilisate product, the enzyme was dialyzed the day before analysis. 200 mg of protein dissolved in 2 ml of 0.2 M Tris-HCI (pll 7 5) contaimng 5 mM CaCi,, were dialyzed overnight agmnst 2 I of the san,e buffer. After dialysis the enzyme ~ l u tion was br,,ul,~:: to 4 ml with the same buffer. Leucine an mopeptidase. Leucine aminopeptidase was purchased fr¢ m Sigma as suspension in 2 m g / m l ammonium suipk ate. To avoid interference by ammonium ions for the amino acid analyses, the enzyme was prepared in glyce:oi solution. 5 ml of enzyme suspensiou were centrifuged at 30000 x g for 5-10 rain and the precipitate was dissolved in 1 ml of a solu:'on consisting of 50% glycerol in 0.1 M phosphate buffer (pH 7.0) containing 5 mtvl MgCl:. The final concentration of enzyme was 10 m g / m l ~750-1500 U/ml). D-Amino acid oxidase. D-Amino-acid oxidase was purchased as a ,=~stalline suspension in ammonium sulphate at 5 m g / n d (75 U/ml). To avoid interference by ammonium ions for the amino acids analyses, the enzyme was prepared as follows: 1 ml of enzyme was centrifuged at 300(0 × g for 5-10 rain. The supernatant was discarded and :he precipitate was dissolved in 1 ml of a solution con'~sling of 50c/, glycerol in 0.1 M phosphate buffer (p,' I ?.13). o-Asparrate oxidase (EC 1.4.3.1). This enzyme from Octopus vulgaris wa~ purified according the procedure of D'AnieLIo et al. [7] and stored at 2-5 °C at a con-
centrafion of 10 mg/ml (100 U / m l ) in a solution consisting of 50ff glycerol 0.1 M phosphate buffer (pH
7.0). PeptidyI-D-amino-acidi hvdrola~e/rom L. ~'ulgarix "l'tris was purified according the procedure of D'Aniello etal. [61 and st~red at 2 - 5 ° C at the concentration of 5.7 mg/ml in a solutio, consisting of 50~ glycerol in 0.1 M phosphate buffer (pH 7.0). A mino acid analyses Total amino acids. Were determined according to Doy's procedure method C [8]. 1;,dwidual amino acias. Were determined on an automatic amino acid analyzer (Liquimat 111, Kontron, Zurich. Switzerland). t-Aspartw acid and t-alanine. Were determined using an enzymatic method [9,10]. Preparatwe separation of amino acids. This technique was performed after total hydrolysis of the sample to ~t~aratc each amino acid and thus to determine the amount of D and t-forms. Amino acids from hydrolysates were purified by ion-exchange chromatography as follow: 25 btl of the sample hydrolyzed, were loaded or.to the column of the automatic amino acid analyzer and chromatographed accordi,g to the standard program for protein hydrolysates. The effluent was collected on 0.4 ml fractions. The anuno acids in the fractions were located by spot tests on thin-layer plates, (from each fraction 5 ~1). and visualized by spraying wiwh ninhydrin-collidine reagent. Corresl~onding fractions from five chromatograph!c runs v,ere pooled, to give at least 500 nmol of each amino acid. The pooled fractions were concentrated t,y Jyopluitzation to 0.5 ml. Determination of o-amino acids Spectrophotometric uhraetolet assay of D-asptlrtic acid and D-alanine. These two amino acids were determined specifically by oxidation with D-aspart'~te oxidase and o-amino-acid oxidase, respectively, according to a spectrophotometric enzymatic assay previously described by D'Aniello etal. [7]. To determine D-aspartic acid, 500/~i of sample were adjusted to pH 8.5 with 2 M Tris and incubated with 20 t~l D-aspartate oxidase (2 U), 5 /~1 catalase (2500 U/ml), 0.1 /tmoi ~-NADH, 5 ~.1 malate dehydrogenase (30 U) and 0.1 M "i'ris-HCI (pH 8 5 ) was added to a volume of 1 ml. After 60 rain at 37"C the decrement in absorption at 340 nm was measured against H zO. A blank was carried out in the absence of I>aspartate oxida:~ to measure the spontaneous decrease of fl-NADH. ~Alanine was determined in the same way but Daspartate oxidase and malate dehydrogenase were replaced by 10 pl of D-amino acid oxidase 11.5 U) and 5 ~.1 of lactate dehydrogenase (25 U). A standard curve consisting of various amounts of D-aspartic acid or
202 o-alanine wa~, al~o conMructed undtrm tllc ~,,~me COhOSlions. The sensitivity of this meth~xl is such that reliable measurements can be made with as little a.~ 2.5 nmol of D-3spartic acid or o-alanine in 1 ml of assay mixture eve~ if the same mixture contains up to 30 ~tmoi of the corresponding t-amino acids ,~r of a mixture of t-amino acids (ratio % of DAsp or PAin/total amino acids = 0.0083). Fiuorimetric assay of D-amino acids. This method was used to determine, with high ~nsitivity, the total Damino acids present in a sample after total hydrolysis of proteins. The same method was also used to determine specifically the percentages of racemization of each of the following amino acids: Asp, Giu. Aia, Leu, lie, Val, Phe. Ser. Met and Tyr, obtained after preparative ionexchange column chromatography (see above). The method exploits the finding of the Gross et al. [11] that H,O., produced by the reaction between a D-amino acid and D-amino-acid oxidase or D-aspartate oxidase produce with tyramine a highly fluorescent biphenyl derivative. 0.1-0.5 ml of sample or each amino zcid purified, was adjusted to pH 8.5 with 2 M Tris and mixed with 0.5 mi of 3 mM tyramine (solution in 0.1 M Tris-HCI, pH 8.5), 2 ~1 of perox,idase (25 U) and 0.l M Tris-HCi (pH 8.51 in a final volume of 1.5 ml. The initial fluorescence was read on a spectrofl,~orimeter (Turner model a40), excitation wavelength 325 nm and emission wavleength 415 nm, against a blank consisting of the same assay mixtt, re, hut H :O replaced the sample. The reaction was started by addition of 10 ~1 of D-aspartate oxidase or 10 ~tl of o-aaaino-acid oxidase to the assay mixture. Afler incubation at 37°C for 60 rnin, the sample was cooled t',~ room temperature and the fluorescence was read again. A calibration curve was obtained using a standard consisting of P-aspartic acid or -alanine at a concentration between 0.05 and 5 nmol. F,>r ',he determination of D-aspartic acid and '-)glutamic acid. Oct~pus D-aspartate oxidase was used. since this enzyme o:~idizes only tyhes~ two amino acids [7,121. To determine the following amino acids: PAin. oLeu Dlle. oVal, t)Phe, DPro, oSer. DMct and DTyr hog kidney O-arrdao acid oxidase was used, since it oxidizes the,:e amino acids, but not l)A~p and DGlu [13]. Wl',en this method was used to determine total Damino acids in the whole hydrolysate, we were able to deterrmne as little as 0.5 nmol of total D-amino acids/1.5 mi of assay mixture in the presence of 20 ~mol of a mixture of total L-amino acids (sensitivity D-amino acids/total L-amino acids = 0.000025). When the procedure was used to determine the pelcentage of rzcemization of each amino acid purified on the ion-exchange column, we were able to determine as little as 0.05 nmol of a o-amino acid in the presence of 500 nmol of the corresponding "-amino acid. In this case, the sensibility
expressed as the ratio of D-amino acid/total amino acid = 0.001 that corresponding to a ratio~ of racemization of I ) / D + t a,oino acid = 0.01. Gas chromatography. This was used to determine DArg. DCys, DThr and DLys. since neither t~aspartate oxidase nor D-an-,im-acid oxidase oxidizes them. A Chirasil-tVal capillay column (Chrompak, Middeiburg, The Netherlan.ls) was used and the analysis was carried out on a gas chromatograph, Model Mega 5160 (Carlo Erba, Milano, Italy), according to the method of Frank et al. 1141.
Hydrolysis o/proteins with strongly reduced racemization of amino acids (present method) ~tep i. Partial chemical hydroO'sis. About 10 mg of purified l~roteins dissolved in 1 ml H : O or ! ml of a suspension of a tissue homogenate with a protemn content of about 10 mg/mi, were mixed with 5 ml of 12 M HCI and 100 ~1 of l[]-mercaptoethanol. The tube was sealed off under vacuum and incubated at 37°C for 12 days. The HCI was removed in a rotating evaporator at 4 0 - 5 0 ° C or it was evaporated from Petri dishes on a hot plate at 4 0 - 5 0 ° C in a hood. The dry residue was dissolved in 5 ml 0.1 M Tris-HCI (pH 7.5) containing 5 mM CaCI 2. To check total recovery, a known amount of norleucine was added as internal standard to the sample b,ffore hydrolysis. Step 2. En',ymati: hydrolysis with pronase. The sample was adjusted to p H 7.5 with 2 M Tris and insoluble non-proteic residues were removed by centrifugation at 30000 x g for 60 rain. The supernatant was incubated with 50 ~tl o,~ pronase, prepared as described above and incubated for 12 h at 370C. The pronase was then removed by centrifugation of the sample through a CF 25A Amicon Centriflo membrane cone (cut-off 25000 Daitons) at 3000 x g. After total filtration, 5 mi 0.05 M Tris-HCl (pH ~.5) were centrifuged through the cones for complete recovery of the sample.
Step 3. Enzymatic hydrolysis with ieucine c'ninopeptiduse and peptidyI-D-amino acid hydrolase. The sample was adjusted to pH 8.5 with 2 M Tris and 50 /ai of 1 M MgCI:, 50 btl of leucine aminopeptidase (10 m g / m l ) and 200 tal of peptidyl-o-amino-acid hydrolase (8000 enzymatic units) were added. The solution was incubated for 12 h at 370C. After that, the sample was filtered through CF 25A Amicon Centriflo membrane cones, as in the previous step, to eliminate the hydrolytic enzymes, and finally the filtrate was analyzed for total L- and D-amino acid content. Note. A blank of the proteolytic enzymes was prepared to check the amount of amino acids hydro~yzed by the proteinases themselves, using only the enzymes without sample and distilled water instead of sample. The amino acids released by the proteinases were then subtracted from those obtained after hydrolysis of the
203 sample (about 5-8c[ of total amino acids hydrolyzed from the sample). The degree of total hydrolysis of the proteins after each procedure step was compared with that obtained from a sample which was duplicate except that it was hydrolyzed in 6 M HCI at I 1 0 ° C for 24 h (total chemical hydrolysis). It should be no',ed that the amino acids released are due to hydrolysis of the added proteinases. This control assumes that autodigestion of the proteinases occurs at the same rate in the presence and absence of added
minimum level. For this stud), human serum was chosen as a sample of protein mixture. Attention was focused on the racemization of aspartic acid and alanine. since the first racemizes at high rat ; with respect to other amino acids, whereas the second represents a medium value. Furthermore. for these two amino acids a specific enzymatic analysis is available for the D form as well as for the t form. When samples of human serum 0 5 0 ~! containing about 10 mg protein) were subjected to hydrolysis in 6 M and 10 M HCi at temperatures ranging from 1 0 o c to l l 0 ° C for 24 h, the rate of racemizafion was not appreciable up to 40°C, whereas above this value it increased exponentially with temperature (Fig. 1). in 10 M HC! for 24 h at 750C the racemized aspartic acid is about 1.4,% (expressed as the ratio D/D + t amino acid x 100), while at I I 0 ° C it is about 6.5%. The corresponding values for alanine were 0.5~ at 75°C and 1.2~ at l l 0 ° C . Because of the extremely low racemization occurring below a 0 ° C , the rate of hydrolysis along with the rate of racemization of aspartic acid were determined during incubation of the human serum for several days in 6 M or 10 M HCI at 37°C. Fig. IB shows the results of ,his study. It is noteworthy that after 12 days of incubation at 37°C, the diff~ence of the rate of hydrolysis of proteins in 6 M and 10 M HCI is about 20% wit.h respect to total hydrolysis. However, the percent of racemization of asparfc acid was only 0.015% expressed as the ratio% of D / D + t (Fig. 1).
substrates.
llemlts Effect of temperature, time and acid con<'ent.vation nn the rate of hydrolysis and on racemization of amino acids in initial attempts to obtain total enzymatic hydrolysis of proteins, and the liberation of the D-amino acids. we used various endo- and exopeptidases (papain. pepsin, chymotrypsin A, neutral proteinase, pronase. carboxipeptidase A and leucine aminopeptidase), following the optimal assay conditions given for each hydrolase (see respective Refs. 15-21), coupling with peptidyI-D-amino-acid hydrolase. Unfortunately. using this procedure we were unable to obtain complete hydrolysis of the proteins, since this last enzyme can split peptide bonds of peptides composed of not more than five residues of D- or L-amino acids [6]. Therefore. it seemed necessary to split the protein chain to fragments by chemical means before the enzymatic completion of hydrolysis. A preliminary study was performed in order to establish the conditions of partial chemical hydrolysis in which the racemization of amino acids occurs at a
Rate of hydro/ysis o/proteins and comparative study of the racemization of the amino acids Fig. 2 shows a typical profile of the sequential procedure for complete hydrolysis of proteins. The results are the mean values of four hydrolyses of a crude homo-
iI" ?
E
~
0/
!,
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3
~3o
Io
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'
lu
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50
70
HyClPolysts t e r n l ~ r a t u r e
90
I~0 " c
4
,
J
8
*
m
=
,
12
t
-"
~s dG),s
Hydro;ys,5 at 37 "C
Fig. I. 'Rate of racemiZatKm of anuno aods in 6 M and 10 M HCI at vanous t i m ~ and temperatures. The sample u.~:l in this expc~mcnt ~a.~ bovine serum albumin. Pand A shows the mean value oi the rate of racemization of ~ c acid and alanin¢ after hydrolysis [or 24 h m 6 M and l0 M HC! at temperatures betwen I0 o C aad 110 o C. Panel B shows the percentage or total hydrolysis ( ) of bovine scrim3 albcum,n at 37 o C in f: M and in 10 M HC1 and the percentage of racemizauon of ~partic acid t . . . . . . ) after hydrotysts of the same protons m the same condmons.
204 I
~
step~
----.~
I
step 2
i
F--
s%ep3
I
-~ - - - - - S 8O % ¢, 6 0
~
to0,
4o
o
~c ~o
$- . . . .
~_
e- . . . .
•
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oL
0
4
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12
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6
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12
Hour5
.....
• . . . .
•
~ ....
4----
0
6
0015
v_
o~
~og
~ 12.
HOURS
Fig. 2. Sequential steps of hydrolysis of proteins with very. low values of amino acid racemization. The values refer to the mean of the results obtained from the following samples of protein: bovmc ~r~nt albumin, human serum, bovine leg muscle. Octop~ vulgaris hepatopancreas and homogenate of peas. - - , total hydrolysi: of proteins; . . . . . . , racen'uzation of the anuno acids. Step 1: hydrolysis in 10 M HC1 at 3 7 ° C . Step 2: enzymatic hydrolysl., with pronas¢. Step 3: enzymatic hydrolysis by leucine aminopeptidase combined with peptid~l-o-amima-acid hydrola~.
genate of bovine leg muscle (taken as an example of a mixture of various kind of proteins). Three stpes are required to obtain total hydrolysis. For the first step, tl'.e chemical hydrolysis, conditions were chosen such as suggested by the data described above, that is. low temperature and long duration of trea.'meat by 10 M HCI. The perc¢ntage of hydrolysis reached after 12 days is about 50%, while th= percentage
of racemization was kept below 0.015% (ratio~ O/D + L) for aspartic acid and 0.01% for alanine. The second step, treatment w i ~ pron~.~e ~'~, b.'5.r,~ :he hydrolysis of proteins from about 50% to about 80%, with respect to total hydrolysis. Finally, the third step completes hydrolys;.s by simultaneous treatment with leucine aminopeptidase and peptidyl-o-amino-acid hydrolase. In the last step we obtained total hydrolysis of pro-
TABLE i
l!,d,~,(, ; ~,f protem.~ bl 16 M HCi fi~r 1S days at 37°C and by enzymatw hydrolysis The values refer to the percent of tot,~l i13,drolysis of the amino acids with respect to that obtained after h_',drolysis in 6 M HCI at i i 0 * C for 24 h. A refers, tc the percent of each amino acid h)'drolyzed after treatment with 10 M HCI at 3 7 ° C for 12 days. B refers to the percent of each amino acid h}dtolyzed alter the complete hydrolysis of proteins [tO M HCI at 37 ° C for 12 days + enzymatic) treatment vnth ptonas¢ and with leu~ane anunopeptiaas¢ together w~th peptidyl-t>-amino-acid hydrolasc. -,,n,, acids
Asp t'hr Ser Glu Pro Gly Aia Va] Cy~ Met I le Leu Tyr Phe Lys !-|i ~ Arg
Bo~,me serum albumin
Human serum
Bovine mu.~le
Hcpatopancreas of
Homogenate
of leg
Octopus t'ulgarls
of pe~s
A
B
A
B
A
B
A
B
A
B
38 39 43 4~ 60 5g 56 41 4g 59 52 54 60 52 59 55 67
100 101 t~ 98 99 ll.X) 101 99 100 101 9g g9 I00 100 10t) I01 98
40 3b 47 46 65 65 46 38 50 61 50 56 73 4q 60 50 70
I00 98 100 100 qq 101 100 gO 99 1(30
45 35 49 50 70 64 48 46 59 55 M
100 ,08 100 9g IOl 100
63 ¢~ 43 54 65 60
51 43 53 4g 68 60 43 49 55 49 41 63 57 44 50 58 61
100 9g 98 97 10(.I 99 101 10l IO3 9g g7 g8 99 100 101 100 100
3~ M 55 54 73 68 44
100
99 98 !00 99 101 i(30 100 99 1(30 98 99 100 100 100 99 9~ 101
45
100 9g 98 101 100 98 100 100
65
100
63 35 62 80 43 62 70 5.5
i01 98 58 100 100 101 97 g8
205 teins with the liberation not only of all L-amino acids. but also of all of D-amino acids if they were present in the sample in examination. In order to verify that a similar pattern was ,)brained also in other proteins, either purified or a mLtture of non-purified proteins, bo~me serum albumir,, human hemoglobin, crude homogenate of octupos hepatopancreas arm crude homogenate of peas. ~ere subjected to the hydnolysis procedure described. As is shown in Table I, column A, in the first step of hydrolysis, the percentage of each amino acid released is about 40-60% ~ t h respect to total acid hydrolysis that occurs at I I 0 * C in 6 M HCI for 24 h (Fig. 1A). After the third step of hydrolysis, all the amino acids composing the proteins under examination, were completely hydrolyzed (Table !. column B). Tal~!e !! shows the percentage of racemization of each amino acid (expressed as the ratio % D/D + LL after total hydrolysis of proteins using the present method in comparison with that obtained when the proteins were hydrolyzed in 6 M HCI at l l 0 ° C for 24 h [I.3-4]. As is evident from the values listed in the same table, using the present method, after total hydrolysis of proteins, aspartic acid r a c e m e s at a percentage that is less than 0.015%, whereas the following amino acids: glutamic acid, alanine, leucine, isoleucine, valine, proline. phenylalanine, serine, methionine and tyrosine racemize less than 0.01%. On the contrary, when the proteins are hydrolyzed at elevated temperature, the
amino acids do racemize at a much higher r~te. In fact. as reported by other investigators [1-4]. under the usual conditions of total chemical hydrolysis of proteins, the amino acids ratmmize between 1 and 9% (ratio D / D + L × 100) depending on the kind of amino acid and the protein treated (Table II. superscripts a. b and c). As for the following amino acids: arginine, cystine, threonine and lysine, since the D-enantiomers of these amino acids are not oxidized by D-aspartate oxidase or by D-amino acid oxidase, they were determined by gas chromatography according to Frank et al. [14]. In ahi~ case, since the minimum value for measuring a ratio of D/L-amino acid should not he below 0.1%, the values of racemiTation for these amino acids are expressed as below 0.I~, (Table II). We were unable to evaluate the rate of racemization of the last two amino acids listed in this table, because tryptophan is destroyed in HCI, and histidine cannot be determined with gas-chromatography techniques [14].
Hydrolysis of synthetic peptides containing D-amino acids and of human cataract lenses Up to this point we have shown that using the present method, total hydrolysis of proteins accompanied by an almost negligible racemization of amino acids can be obtained, in order to prove that the method completely releases the t>-amino acids occurring in peptides or proteins, we used several commercially available synthetic peptides containing D-amino acids
T A B L E II
Comparauve stud~ cm the percentage of raceml'_at~on o/amino actd~ under different conditions o/h~drd)*'ts T h e ~alue~ refer to the percent of racemtzation e~tpres~ed as the r a t i o D / D + t X 100.
Asp Giu Ala Leu lie Val Phe Pro Ser Met Tyr Arg Cys: Thr Lys
a-Lactalhuman J
B-lactal bumnn ~
l]k~vine a plasma albumin
Insuhn"
5.3 1.9 1.2 3.7 37 2.6 1.0
4.9 3.4 1.4
72 3.6 2.. 3.3 4.9 1.1 2.4
5.6 9.~ 1.4 1.9
2.2 I 2 0.9 3.5
5.4
5.1 1.3
1.2
His Trp • b ¢ d
Hychrolysis Hydrolysis H~drolysas Hydrolysis
8.4 2.0
-
0.43 29 0.2 2.2 I.)
-l.l
9.0
Bradyktnm •
2.0
3.85 2.4 0.45 1.6~" -
Bovnne 0 ~,¢rum albumin
Human a hemoglobin
Hcpatopanereas a of Octopus vulgaris
Hom~gcnate a of peas
< 0.015 < 001 < 0.01 < O01 < 0.01 < 0 01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.1 <0.1 <0.l < 0.1
< 0.015 ~ 0.01 < 0.01 < 00! < G.01 < 0.0l < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.1 <0.1 < 01 < 0. I
< < < < < < < < < < < <
< 0.0A5 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 001 < 0.01 < 0.01 < 0.1 <0.l "-0.I < 0. I
for 24 h for 24 h for 22 h in 10 M
in 6 in 6 m 6 HC!
M HCI at l l 0 ° C ( L i a r d o n c t a l . Rcf. 4). M HCI a t l i 0 ° C ( F r a n k c-t al., Rcf. 3; D a t a e x t r a p o l a t e d froth, Fig. 2). M HC1 at l l 0 o C ( M a n n i n g a n d Moore. Rcf. 1). for 12 d a y s at 3 7 ° C followed by e n z y m a t i c hydrol.~s,, ~.p,e,,ent ~ o r k L
0.015 0.01 0.01 0.01 0.01 0.01 0.01 LI.01 0.01 0.01 (I.O~ I:.1
,.0.1 < 0.1 < 0.1 -
2O6 TABLE III Amount of t- and
P-amino
acids hberated from pepndes and protems hr en:ymatrc hrdrv(*,,t.~ fStep ." ond Step 3;
The concentration of amino acids liberated is calculated from the chromatographic analysis before and after ~reatment wilh D-amino-acid o~a~cs¢. The values given in parentheses are the values obtained after oxidas¢ treatment. Anuno acids liberated from 1 pmol of peptide
Synthetic pcptides
T~
DAla
Phe
Leu
IoAla2-Lcul ¢nkcphalin ( Tyr- DAla-Gly-Phc-Leu)
0.98 (0.96
1.03 I (0.96) (0)
0.96 (1.02)
0.98 (I.02)
[oAla:-DLcu s I enkephalinamid¢ ITyr-DAla-Gly- Phe-DLeu • N H ~)
(0.98)
0.98 (099)
0.97 (l.OI)
1.05
0.9~
[[)AIa2-DMeI ~] enkephalin
(Tyr-DAla-Gly- Phe-DMet)
1.02 0.96
! .03
(0) 0.97
(0.98) (0)
Gly
oLeu
DMcI
Tip
DPh¢
Scr
DArt
LArg
Pro
1.98 (2.02)
0.91,l (0)
1.02 (0.99)
0.96 (0.8)
1.02 (1.0)
0.98 (0.(16)
0.95
(0) 0.98 (0)
(0.991 (1.02)
LH-RH hormone
(AoctyI-DTIP-DpCI- Ph¢-DTip-Scr-Tyr-DArg-Leu-Arg-pro-DAla.NH2)
1.03 (0.95)
0.97 ( 1.02 )
1.02
(l.0)
Central nucleus of human lenses cataract Total chemical hydrolysis (6 M HClat I I 0 ° C for 24 h)
Total hydrolysis ~ith
total amino acids (~moi/1O mg of ti~u¢)
total aspartic acid (it tool/10 mg of tissue)
total amano acids ( p m o l / 1 0 mg of tissue)
total DAsp (jamol/10 mg of tissue)
total t.Asp (/~mol/10 mg of tissue1
total Asp (/Amot/10 mg of tisMlel
22.8
2.1
22.1
0.260 (I 2.3c~) ~
1.84
2.1
24.1
2.2
23.,)
0.256 (! 1.9~) .I
1.90
2.15
present method
Women between 71 and 76 years Men between 57 and 72 year~
" Refers to r trio q~ of t)/D + x. aspartic acid.
and human lens cateract, which contains a large amount of D-aspartic acid [22-23]. As peptides containing D-amino acids, we chose the following: [DAia2-1eucine] enkephalin. [oAla 2. DLeu s] enkephalinamide, [DAla:, D M e : ] enkephalin and the LH-RH-luteinizing hormones containing DTrp, DArg and oAla. 1 mg of each of these compounds was dissolved in 1 mi Tris-HCl buffer (pH 7.5) containing 5 mM CaCI,~ and subjected directly ta the two enzymatic hydrolyses (Steps 2 and 3) as described above, but using the amount of the hydrolytic enzymes in proportional quantities. The first step was omitted, since there was no need for a partial hydrolysis, the peptides being already relatively small. After hydrolysis, half the amount of each sample was brought to pH 2.2 and was subjected to amino acid analysis to determine the content of amino acids liberated. The other half was treated with 5 #1 of D-araino-acio ox:.dase and 5 pl of catalase to destroy D-an',ino acids and then analyzed as the first one i7]. The results obtained from this investigation demonstrated that the yield of the L- and D-amino acids was equal to the expected values. In fact, as is shown i,l Table I!I. in which the various pcptides containing D-amino acids after enzymatic hydrolysis using our procedure are reported, all amino acids consisting of the
peptides either in D or L form are completely liberated. From I /~mol of the pcptide DAla'--Leu-enkcphalin we obtained amounts of amino acids near to unity. The same results were obtained when the other three peptides were used (Table III). The eye lens cataracts studied had been sungic',dly removed from four women between 71-76 years of age, and from four men between 57 and 72 years of age. The two groups of four lenses were homogenized in distilled water and were subjected to the hydrolysis procedures as described in Materials and Methods. A portion of same samples were hydrolyzed in 6 M HCI for 24 h at I 1 0 ° C to determine the absolute content of aspartic acid. As is shown in Table !il, using an equal quantity of sample, after our hydrolysis procedure, we found a concentration of total amino acids and of total aspartic acid that was the same as that obtained from chemical hydrolysis (110°C for 24 h in 6 M HCI). Regarding specifically the content of D-aspartic acid in tl'~,c tissues, using our method, we found 0,260 /amo! of Daspartic acid and 1.84 pmoi of L-aspartic acid in 10 mg of the lenses from the women. This value equals 12.3% of the D form. ~ith respect to total amino acid ( D / D . L × 100). Since the sum of DAsp plus LAsp is equal to total aspartic acid found using total chemical hydrolysis
2O7 F----
step 1 - - ~
I
step 2
I
I----- step 3
'°° t
~C
A -112
6(:
O
j
/
/
/
B~s
/
0
0-- . . . .
~oO
C
O'-- - - - - O
v
,q ,
0
i
•
I
8
4
,
I
~2
C
~Gy~
6 ~..:r~
~2
6
12
b4OUrS
Fig. ~. Sequcmial steps of hydrol~'szs of the p¢oteln~, of human cataract lenses. The values refer to the mean of the results of hydrolysis nf eight hmscs. - - . total hydroi)~ts 04" proteins" . . . . . . . racemmation of the amino acids. Step h hydrolysis in 10 M HCI at 3 7 ° C . Step 2: hydro~vsas ~tath p r o t t ~ . Step 3: enzymatic h vdrolyms wtth leucme aminopeptJda.s¢ combined with pept~lyl-o-~mano-acid hydro4ase. In step 3. it~licates, hydrolysis earned out with: (A) Ict~m¢ a m m o p e p l i d a ' ~ and peptld~l-D-ammo acid hydrola.~; (B) I~tcilfl~ amino peptidas¢ only; (C) only with the peptidyl-t~ammo-a~d h~dtol&~. - . . . . . . amount of o-aspartic acid released from pCplidCs after treatment with: (A) |¢ucir~ amtnopepttdas¢ and pcptid),bI>-amlra3-acgt h~,drtdas¢. (B) with only ixq0tidyl-o--ammo-acid hydrolas¢, and (C) with only Ieucin¢ amanopept~das¢.
(6 M HCI, 24 h. I I 0 ° C ) , we conclude that the content of o-aspartic acid, with respect to total aspartic acid, is the real value. Approximately the same results were obtained on cataract lenses from the men (Table Ill). Fig. 3 represents the hydrolysis pattern of the eye lens cataracts. D-Aspartic acid released it. t.t~e first step was about 5~ of the total. It remains at 5~ during hydrolysis with pronase (step 2). The impor,ance of the action of peptidyI-D-aminoacid hydrolase clearly emerges in step 3. In fact, leucine aminopeptidase alone h)drolyzed almost the all L-armno acid (Fig- 3, curve B. unbroken line), but not o-aspartic acid (Fig. 3, curve C, broken line). PeptidyI-D-armnoacid hydrolase alone caused only a small increase of the total hydrolysis of L-amino acids (Fig. 3, C. unbrnken line), but it induced a 5-8c~ release of total o-aspartale present in the proteins (Fig. 3, curve B, broken line). Lastly, peptidyl-t>-amino-acid hydrolase assoc;.z:~ed with leucine aminopeptidase resulted in the total hydrolysis of proteins (Fig- 3, curve A, unbroken line) and total release of D-aspartic acid (Fig- 3, curve A, broken line). l~semskm D-Aspartate was found in the free form in the n e r v o ~ system of Octopus v~dgar~ Sepm officinalis and Loligo vuigar/s [7.24] and i>-.'flani~¢ in Crustacean muscle and hepatopancreas [25], and we hypothesized that this compound is present in proteins or peptides, from which it may be released by a specific peptidase. For these rmsons, we developed a new method of hydrolyzing peptides arm proteins with a very Io,~" degree of amino
acid racemization. This method is parucularly ~uitable for determination of the rate of racemization ol ar,.ino acids in peptides and proteins which can occur in vivo at very low, but still significant levels. A crucial point to obta,n total hydrolysis, is that leucine aminopeptidase and peptidyl-o-amino-acid hydrolase mus: be used together, because if a o-amino acid is present in peptide residues, the leucine aminopeptidase cannot split J',¢ peptide bonds consisting of two o-amino acids or by an L-amino acid and a o-amino acid. Therefore. when a o-amino ac;.~ i~ situat~,.~, in the N-terminal position, only the peptidyI-D-amino-acid hydrolase can cleave the bond. Once o-amino acid is released, the leucine aminopeptidase :ondnues to split the peptide until it meets the next t>-amino acid and so on.
To evaluate the percentage of racerrdzation, we tested aspartic acid. b ~ u a s ¢ it has a higher rate of racemization than other amino acids, and alanine, because it racemizes at a laedium rate a.g compared to other amino acids. Hence. the values obtained for these amino acids reflect the rate of racemization of other amino acids. We -used the enzymatic fluorimetric method instead of gas-chromatography techniques [141 or HPLC techniques [26-27], since it allows reliable determination of the percentage of racemization of amino acids as low as 0.01% (ratio D / D + L X 100). l>-Aspartic acid at high levels has bee~ found iL hur,~an tissues, such as lenses during ageing and in cataract lenses [22,23], tooth enamel [28-29], brain [30-31] and erythrocytes [32]. in all these studies to reduce the deodec of racemization, the proteins were
208
hydrolyzed for only 6 h in HCI at 100°C. However. also under these conditions, a considerable amount of racemization of amino acids took place. In fact. we found that under these hydrolysis conditions, aspartic acid racemizes at a rate between 0.8 and 1.27o (exp:essed as ratio D/L + t X 100), while alan(he racemizes between 0.2 and 0.4% depending on the type of proteins. In the studies mentioned above, aspartic acid racermzation values found in brain of young people were taken as blank values, because the natural racemization in these tissues is very low. The valu~ found in these tissues was subtracted form the values obtained from corresponding tissue from elderly individuals. However. it should be noted first that the ra:e of racemization of amino acid~ induced by chemical hydrolysis is influenced by neighbouring amino acids (Table ll), and second, there is no evidence tha~, the protein composition of the brain of a young person is identical to that of an elderly person. McFadden and Clarke [33] and Geiger and Clarke [34] have report that under some assay conditions of near-neutral pH salues, D-isoaspartate :esidues may arise from some peptides undergoing deamination. Since o-aspartate oxidase which we used does not oxidize such compounds, a small amount of D-aspartic acid may be underestimated. The present method, applied to the proteins and peptides used in this work, leads to total hydrol)sis. Since these compounds comprised pu6fied proteins as either whole animal or vegetable tissues, it is very probable that the method hydrolyzes all proteins. However, the amount of total amino acids obtained with the present method should be compared vdth that obtained using the classic method of chemical hydrolysis (6 M HCI at 110 o C for 24 h). In conclusion, the method described in this paper greatly reduces racemization of amino acids during the hydrolysis of proteins, h is suitable for the hydrolysis of peptides and proteins consisting not only of L-amino acids, but also of I>-atnino acids. It is lengthy compared t,~ no.qnal acid hydrolysis, but it is the only procedure yet available that permits the detection of small amounts of racemization of amino acids in vivo that wot.ld otherwise have been inde~.ectable. Acknowledgements We are grateful to Dr. Giovanna Nard( and Professor J. Manuel Denuc6 for critical examination of the manuscript. We also thank Mrs. Giuseppa Trovaglione of the Department of Biological Chemistry. of the University of Naples for performing amino acid analyses and Professor F. Menna and co-workecs, of Pellegrini Hospital in Naples. for providing human cataract lenses.
References 1 Manning. ,'.M and Moore, S (1968) J. Biol Chem. 243. 5591-5597. 2 Manning..;.M. (1970) J. Am. Chem. Yroc.92, 7449-7454 3 Frank, H.. Wolfg~ng. W.. NichoLson. G. and Ba,'et. E (1981) Liebigs Ann. Chem. 354-365. 4 Liardon, R., Lederman, S. and Oil. U. l19811J. Chromatogr. 203. 385-395. 5 Sletten. K., Dus, K., De Klerk, H and Kamen. M.D. 0968) J. Biol. Chem. 243. 5492-5506. 6 D'Aniello. A. and Strazzullo, L (1984) J. Biol. Chem. 259. 4237-4243. 7 D'Aniello. A. and Giudttta, A. (1977) J. Neure~hent 29. 0.53-1057. 8 Doy, I~, Shlbata, D. and Matoba, ]['. (1981) Anal. B ~ h e m . It ', 173-194. 9 Bergmeyer. H.U.. Berm. E., Mollermg. M. and ~ e r . G. (1974) in Methods of EJnz)mauc Analysts (Bc-rgmay~. H.U., od.k Vol. 4, pp. 1696-I"700. Academic Press. New York. l0 Williamson. D.H. (1974) in Methods ¢ff Enzymat!: A,:-Iysis (Bergrnay©r, H.U.. ¢d.). Vol. 4, pp. i(~79-1685. Ac~ka'm¢ Press. New York. 11 Gros,s, A.T. and Sirer, I.W. (1959) J. Biol. Churn 234, 1611-1614. 12 D'Aniello, A. and Rocca. E. (1972) Cutup. Biochcm. Physiol. 4lB, 625 -633, 13 Dixson. M. and Kleppe. K. (1065) Biachim. Ikophys. Acta 96. 368-382. 14 Frank, H.. Graem¢, J.N. and Ba)er, E. (1978)J. Chromatogr. 167. 187-196. 15 Bcrgmcy~. H.U., Gawdm. K. and Gros,sl, M. (1974) in Nk-thods of Enzymatic Analysts (Bcrgm~et. H.U.. ¢d.kVol. I. pp. 491-492. Academic Press. New Yo~. 16 Bergmeyer. H.U., Gawehn. K. and Gross.. M. (1974) in Methods of Enzymatic Ana*ysis ( l ~ ¢ r . H.U. ed.L VoL I, pp. 493--494, Academic Pl~_,s. New York. 17 Bergmcye;. H.U.. Gawehm K. and G ossl, M. (1974) in Methods of Enzymatic Analysis (Bcq~neycr. P U., ed.), VoL i. pp. 440- IA?. Academic Press. New York. 15 Yasunobu. K.T. and MOConm J. (b'vO) Methods Em,ymoL 19. 569-575. 19 Naraha.shi. Y. (1970) Methods Enz)ax~l. '9. 651-664 20 Appel. W. (1974) in Methods of Enzywaluc ~,nalysas (Ik.rgn~)er. HU.. ed.L VOl. 2, pp. 954-964. Academic Pre,, N,,~. Y,xrk. 22 Master,, P.M.. Bad~ J.L. and Zi~.ler, J.S, Jr. (tOT/) Nature 268. 71-73. 23 Mastet,~, P.M., P,ada, JL. and Zigler. J.S.. Jr. (1978) Proc. Natl. Acad. S¢*. USA 75. 1.-~4-1208. 24 D'Anie(|e. A. and Giuditta, A. (1978) J. Neurochem. 31. 1107-1108. 23 D'Aniello, A. and Giuditta. A. (1980) Comp, Biod~m. Physiol. 66B. 31g-322. 26 Dunlop, DS. and N,:idle. A. (1987) Anal. Biochem. 165.38-44. 28 Helfman. P.M. and F.;ada. J.L. (1975) Proc. Natl. Acad. So. USA 72. 2897-2894. 29 ltelfman. P.M. and Bada, J.L (1976) Nature (London), 262. 279-2gl. 30 Man. F.lt., Sandhouse, M.F., Burg, .I and F:sher. G.H. (1983) Science 220, 1407-1408. 31 Man. F,H. et al. (1987~ J. Neurochem. 48. 510-515. 32 Bruna~cr, L. and Clarke. S. (1986) J. Biol. Chem 261, 12538-12543. 33 McFadden. P.N. and Clarke, S. (1987) Ptoc. Natl. Acid. Sci. USA 34 Geiger. T. and Clarke. S. 0987) J. Biol. Chem. 262. 785-794.
Btochmaca el Btophvswa Acta, 1037 (19qO) 200-208 El.~evaer
B~APRO 33544
Total hydrolysis of proteins with strongly reduced racemization of amino acids Antimo D'Aniello, Guiseppe D'Onofrio and Michele Pischetola Department of fliochemlst~,, Sla~lone Zoologwa "A Do~rn : Vdla Comunale, Naples (Italy') (R~et~ed 27 June 1999)
Key gords: Attune acids racemw.ation: D-Aspartate: D-Amino acid: Protein hydrolysis; D-Amino aod hydrolysis; (Human cata:~l lens)
A new method has been devised for the complete hydrolysis oi pcoteins with an extremely low level of racem/z'z~i~a ol amino acids. Proteins are incubated in 10 M HCI at • low temperature to obtain partial hydl~lysis, qIhey are then incubated with pconase and finally with leucine a m i ~ and peptidybD-amino-acid hydrolnse from vu/~m'& The proposed method ensures the total hydrolysis of either puriCM~dpcoteins or protei~ contained in • crude homogenate of animal or vegetable tissue. In both cases, the racemization of ,.alao acids (expressed as rate d D f o r m / o + L form X 100) was lower than 0.015% for aspartic acid and lower than 0.01% for other amino acids. D-Amino acids released from peptides or proteins were estimated with enzymatic methods based on the use of octopus D-aspartate oxidase or hog kidney D-amino acid oxidase; with tlbe~ e n z y m ~ 0.05 nmoi o~ • D-am/tao acid was d~ecmimsd in the presence of up to 20 /~mol of a mixture ol L-amino acids (ratio % D / D + L =0.00025). The met4md allows ~ e determination of l>-amino acids either in tissues in which they are present in high ctmcentratioas (as human cataract lenses, tooth enamel, etc.) or in those with low emmtlomer content (as brain erythrocytes, etc.). Using the described, we hydrolyzed several synthetic peptides consisting el i>. and L-amino acids and d e t e n n i n ~ the a m m ~ d D-amino acids. In addition, we totally hydrolyzed all the nuclear proteins of human eataractous lenses. The amount og D-aspartic acid was 0.026 lamoi/mg in lenses of women aged between 71 and 76 years and 0.07~6/zmol/mg in lenses of men aged between 55 and 72 years. The D-aspartlc acid measured corresponds to about 12% with respect to total aspartie acid, Introduction The purpose of the present work was to determine the content o1 D-amino acids in pzptides and proteins to a high level of accuracy. A preliminary step of ',his analysis is to completely hydrolyzc these compounds to obtain free amine adds from which the D-amino acids levels can then he determined. The chemical metho.'i normally used to hydrolyze proteins (6 M HCI at ~10 °C for 24 h) provokes racemization of amino acids [1-4]; and enzymatic hydrolysis, which does not cause racemization of amino acids, does not bring about total hydrolysis of proteins. As far as we are aware, apart from the complete hydrolysis of cytochrome c [5], there is no other example of complete hydrolysis of proteins using only hydrolases. In ad-
Correspondence: A. D'Anidlo, Stazion¢ Zo~loglca "A. Dohm', Villa Comunale, 80121 Napoli, Italy.
difon, no one hydrolytic enzyme yet studied is able to cut peptide bonds consisting of D-amino acids. In a previous study, we purified peptidyi-l>-aminoacid hydrolase from the intestine of the Loligo t,ulgaris that hydrolyzes small peptides consisting of D- or Lamino acids [6]. This prompted us to study whether rids enzyme could be used to free D-amino acids from their proteic compounds. In this work we describe a new method for t~he complete hydrolysis of proteins either in a purified form or contained in whole animal and vegetable tissues which concomitantly strongly reduces racemization of amino acids. This method is carried out firstly by chemical hydrolysis in I0 M HCI at a low temperature to obtain a small proteic fragment and then continuing hydrolysis using, pronase. Finally, leucine aminopeptida~ with peptidyl-l>-amino-acid hydrolase complete the hydrolysis. Using this procedure we were able to determine precisely the D-amino acid content in various peptides and proteins. These results induced us to conclude that
016%4838/90/,933.50 ~: 1990 Elsm'ier Science Pubhshers B.V. ~,Biomcdical Dies,on)
201 the present method may be considered as a general procedure to determine D-amino acids in proteic co,mpounds.
Materials and Methods Chemicals Peroxidase from horseradish (250 U/mg. 10 mg/mi, suspension), malate dehydrogenase (1200 U / m g . 10 mg/ml, solution in glycerol, 50%), lactate dehydrogenase (550 U / m g , 10 mg/ml, solution in glycerol, 50%), papain (EC 3A.22.2), chymotrypsin A4 (EC 3.4.21.1), tryosin (EC 3.4.21.4). neutral proteinase (EC 3.4.24.4), pronase from S. griseus, carboxypeptidase (EC 3.4.12.1), D-amino-acid oxidase (EC 1.4.3.3) from hog kidney, and reduced ~-nicotinamideadenine dinucleotide (/]-NADH) disodium salt. were purchased from Boehringen Mannheim. Uhrafihration membrane cones type CF 25 were from Amicon, U.S.A. Leucine aminopeptidase (EC 3.4.1 !.1 ). peroxidase (EC 1.11.1.7) from horseradish, 10 m g / m i (2500 U/ml). catalase (EC 1.11.1.6) from bovine liver 15 mg/ml (50000 U/rag) and all other chemical used in this work were from Sigma. St. Louis. MO, U.S.A. En:yme preparations Pronase (approx. 7000 U / g tyophilisate). In order to eliminate possible contamination by free amino acids or peptides contained in the lyophilisate product, the enzyme was dialyzed the day before analysis. 200 mg of protein dissolved in 2 ml of 0.2 M Tris-HCI (pll 7 5) contaimng 5 mM CaCi,, were dialyzed overnight agmnst 2 I of the san,e buffer. After dialysis the enzyme ~ l u tion was br,,ul,~:: to 4 ml with the same buffer. Leucine an mopeptidase. Leucine aminopeptidase was purchased fr¢ m Sigma as suspension in 2 m g / m l ammonium suipk ate. To avoid interference by ammonium ions for the amino acid analyses, the enzyme was prepared in glyce:oi solution. 5 ml of enzyme suspensiou were centrifuged at 30000 x g for 5-10 rain and the precipitate was dissolved in 1 ml of a solu:'on consisting of 50% glycerol in 0.1 M phosphate buffer (pH 7.0) containing 5 mtvl MgCl:. The final concentration of enzyme was 10 m g / m l ~750-1500 U/ml). D-Amino acid oxidase. D-Amino-acid oxidase was purchased as a ,=~stalline suspension in ammonium sulphate at 5 m g / n d (75 U/ml). To avoid interference by ammonium ions for the amino acids analyses, the enzyme was prepared as follows: 1 ml of enzyme was centrifuged at 300(0 × g for 5-10 rain. The supernatant was discarded and :he precipitate was dissolved in 1 ml of a solution con'~sling of 50c/, glycerol in 0.1 M phosphate buffer (p,' I ?.13). o-Asparrate oxidase (EC 1.4.3.1). This enzyme from Octopus vulgaris wa~ purified according the procedure of D'AnieLIo et al. [7] and stored at 2-5 °C at a con-
centrafion of 10 mg/ml (100 U / m l ) in a solution consisting of 50ff glycerol 0.1 M phosphate buffer (pH
7.0). PeptidyI-D-amino-acidi hvdrola~e/rom L. ~'ulgarix "l'tris was purified according the procedure of D'Aniello etal. [61 and st~red at 2 - 5 ° C at the concentration of 5.7 mg/ml in a solutio, consisting of 50~ glycerol in 0.1 M phosphate buffer (pH 7.0). A mino acid analyses Total amino acids. Were determined according to Doy's procedure method C [8]. 1;,dwidual amino acias. Were determined on an automatic amino acid analyzer (Liquimat 111, Kontron, Zurich. Switzerland). t-Aspartw acid and t-alanine. Were determined using an enzymatic method [9,10]. Preparatwe separation of amino acids. This technique was performed after total hydrolysis of the sample to ~t~aratc each amino acid and thus to determine the amount of D and t-forms. Amino acids from hydrolysates were purified by ion-exchange chromatography as follow: 25 btl of the sample hydrolyzed, were loaded or.to the column of the automatic amino acid analyzer and chromatographed accordi,g to the standard program for protein hydrolysates. The effluent was collected on 0.4 ml fractions. The anuno acids in the fractions were located by spot tests on thin-layer plates, (from each fraction 5 ~1). and visualized by spraying wiwh ninhydrin-collidine reagent. Corresl~onding fractions from five chromatograph!c runs v,ere pooled, to give at least 500 nmol of each amino acid. The pooled fractions were concentrated t,y Jyopluitzation to 0.5 ml. Determination of o-amino acids Spectrophotometric uhraetolet assay of D-asptlrtic acid and D-alanine. These two amino acids were determined specifically by oxidation with D-aspart'~te oxidase and o-amino-acid oxidase, respectively, according to a spectrophotometric enzymatic assay previously described by D'Aniello etal. [7]. To determine D-aspartic acid, 500/~i of sample were adjusted to pH 8.5 with 2 M Tris and incubated with 20 t~l D-aspartate oxidase (2 U), 5 /~1 catalase (2500 U/ml), 0.1 /tmoi ~-NADH, 5 ~.1 malate dehydrogenase (30 U) and 0.1 M "i'ris-HCI (pH 8 5 ) was added to a volume of 1 ml. After 60 rain at 37"C the decrement in absorption at 340 nm was measured against H zO. A blank was carried out in the absence of I>aspartate oxida:~ to measure the spontaneous decrease of fl-NADH. ~Alanine was determined in the same way but Daspartate oxidase and malate dehydrogenase were replaced by 10 pl of D-amino acid oxidase 11.5 U) and 5 ~.1 of lactate dehydrogenase (25 U). A standard curve consisting of various amounts of D-aspartic acid or
202 o-alanine wa~, al~o conMructed undtrm tllc ~,,~me COhOSlions. The sensitivity of this meth~xl is such that reliable measurements can be made with as little a.~ 2.5 nmol of D-3spartic acid or o-alanine in 1 ml of assay mixture eve~ if the same mixture contains up to 30 ~tmoi of the corresponding t-amino acids ,~r of a mixture of t-amino acids (ratio % of DAsp or PAin/total amino acids = 0.0083). Fiuorimetric assay of D-amino acids. This method was used to determine, with high ~nsitivity, the total Damino acids present in a sample after total hydrolysis of proteins. The same method was also used to determine specifically the percentages of racemization of each of the following amino acids: Asp, Giu. Aia, Leu, lie, Val, Phe. Ser. Met and Tyr, obtained after preparative ionexchange column chromatography (see above). The method exploits the finding of the Gross et al. [11] that H,O., produced by the reaction between a D-amino acid and D-amino-acid oxidase or D-aspartate oxidase produce with tyramine a highly fluorescent biphenyl derivative. 0.1-0.5 ml of sample or each amino zcid purified, was adjusted to pH 8.5 with 2 M Tris and mixed with 0.5 mi of 3 mM tyramine (solution in 0.1 M Tris-HCI, pH 8.5), 2 ~1 of perox,idase (25 U) and 0.l M Tris-HCi (pH 8.51 in a final volume of 1.5 ml. The initial fluorescence was read on a spectrofl,~orimeter (Turner model a40), excitation wavelength 325 nm and emission wavleength 415 nm, against a blank consisting of the same assay mixtt, re, hut H :O replaced the sample. The reaction was started by addition of 10 ~1 of D-aspartate oxidase or 10 ~tl of o-aaaino-acid oxidase to the assay mixture. Afler incubation at 37°C for 60 rnin, the sample was cooled t',~ room temperature and the fluorescence was read again. A calibration curve was obtained using a standard consisting of P-aspartic acid or -alanine at a concentration between 0.05 and 5 nmol. F,>r ',he determination of D-aspartic acid and '-)glutamic acid. Oct~pus D-aspartate oxidase was used. since this enzyme o:~idizes only tyhes~ two amino acids [7,121. To determine the following amino acids: PAin. oLeu Dlle. oVal, t)Phe, DPro, oSer. DMct and DTyr hog kidney O-arrdao acid oxidase was used, since it oxidizes the,:e amino acids, but not l)A~p and DGlu [13]. Wl',en this method was used to determine total Damino acids in the whole hydrolysate, we were able to deterrmne as little as 0.5 nmol of total D-amino acids/1.5 mi of assay mixture in the presence of 20 ~mol of a mixture of total L-amino acids (sensitivity D-amino acids/total L-amino acids = 0.000025). When the procedure was used to determine the pelcentage of rzcemization of each amino acid purified on the ion-exchange column, we were able to determine as little as 0.05 nmol of a o-amino acid in the presence of 500 nmol of the corresponding "-amino acid. In this case, the sensibility
expressed as the ratio of D-amino acid/total amino acid = 0.001 that corresponding to a ratio~ of racemization of I ) / D + t a,oino acid = 0.01. Gas chromatography. This was used to determine DArg. DCys, DThr and DLys. since neither t~aspartate oxidase nor D-an-,im-acid oxidase oxidizes them. A Chirasil-tVal capillay column (Chrompak, Middeiburg, The Netherlan.ls) was used and the analysis was carried out on a gas chromatograph, Model Mega 5160 (Carlo Erba, Milano, Italy), according to the method of Frank et al. 1141.
Hydrolysis o/proteins with strongly reduced racemization of amino acids (present method) ~tep i. Partial chemical hydroO'sis. About 10 mg of purified l~roteins dissolved in 1 ml H : O or ! ml of a suspension of a tissue homogenate with a protemn content of about 10 mg/mi, were mixed with 5 ml of 12 M HCI and 100 ~1 of l[]-mercaptoethanol. The tube was sealed off under vacuum and incubated at 37°C for 12 days. The HCI was removed in a rotating evaporator at 4 0 - 5 0 ° C or it was evaporated from Petri dishes on a hot plate at 4 0 - 5 0 ° C in a hood. The dry residue was dissolved in 5 ml 0.1 M Tris-HCI (pH 7.5) containing 5 mM CaCI 2. To check total recovery, a known amount of norleucine was added as internal standard to the sample b,ffore hydrolysis. Step 2. En',ymati: hydrolysis with pronase. The sample was adjusted to p H 7.5 with 2 M Tris and insoluble non-proteic residues were removed by centrifugation at 30000 x g for 60 rain. The supernatant was incubated with 50 ~tl o,~ pronase, prepared as described above and incubated for 12 h at 370C. The pronase was then removed by centrifugation of the sample through a CF 25A Amicon Centriflo membrane cone (cut-off 25000 Daitons) at 3000 x g. After total filtration, 5 mi 0.05 M Tris-HCl (pH ~.5) were centrifuged through the cones for complete recovery of the sample.
Step 3. Enzymatic hydrolysis with ieucine c'ninopeptiduse and peptidyI-D-amino acid hydrolase. The sample was adjusted to pH 8.5 with 2 M Tris and 50 /ai of 1 M MgCI:, 50 btl of leucine aminopeptidase (10 m g / m l ) and 200 tal of peptidyl-o-amino-acid hydrolase (8000 enzymatic units) were added. The solution was incubated for 12 h at 370C. After that, the sample was filtered through CF 25A Amicon Centriflo membrane cones, as in the previous step, to eliminate the hydrolytic enzymes, and finally the filtrate was analyzed for total L- and D-amino acid content. Note. A blank of the proteolytic enzymes was prepared to check the amount of amino acids hydro~yzed by the proteinases themselves, using only the enzymes without sample and distilled water instead of sample. The amino acids released by the proteinases were then subtracted from those obtained after hydrolysis of the
203 sample (about 5-8c[ of total amino acids hydrolyzed from the sample). The degree of total hydrolysis of the proteins after each procedure step was compared with that obtained from a sample which was duplicate except that it was hydrolyzed in 6 M HCI at I 1 0 ° C for 24 h (total chemical hydrolysis). It should be no',ed that the amino acids released are due to hydrolysis of the added proteinases. This control assumes that autodigestion of the proteinases occurs at the same rate in the presence and absence of added
minimum level. For this stud), human serum was chosen as a sample of protein mixture. Attention was focused on the racemization of aspartic acid and alanine. since the first racemizes at high rat ; with respect to other amino acids, whereas the second represents a medium value. Furthermore. for these two amino acids a specific enzymatic analysis is available for the D form as well as for the t form. When samples of human serum 0 5 0 ~! containing about 10 mg protein) were subjected to hydrolysis in 6 M and 10 M HCi at temperatures ranging from 1 0 o c to l l 0 ° C for 24 h, the rate of racemizafion was not appreciable up to 40°C, whereas above this value it increased exponentially with temperature (Fig. 1). in 10 M HC! for 24 h at 750C the racemized aspartic acid is about 1.4,% (expressed as the ratio D/D + t amino acid x 100), while at I I 0 ° C it is about 6.5%. The corresponding values for alanine were 0.5~ at 75°C and 1.2~ at l l 0 ° C . Because of the extremely low racemization occurring below a 0 ° C , the rate of hydrolysis along with the rate of racemization of aspartic acid were determined during incubation of the human serum for several days in 6 M or 10 M HCI at 37°C. Fig. IB shows the results of ,his study. It is noteworthy that after 12 days of incubation at 37°C, the diff~ence of the rate of hydrolysis of proteins in 6 M and 10 M HCI is about 20% wit.h respect to total hydrolysis. However, the percent of racemization of asparfc acid was only 0.015% expressed as the ratio% of D / D + t (Fig. 1).
substrates.
llemlts Effect of temperature, time and acid con<'ent.vation nn the rate of hydrolysis and on racemization of amino acids in initial attempts to obtain total enzymatic hydrolysis of proteins, and the liberation of the D-amino acids. we used various endo- and exopeptidases (papain. pepsin, chymotrypsin A, neutral proteinase, pronase. carboxipeptidase A and leucine aminopeptidase), following the optimal assay conditions given for each hydrolase (see respective Refs. 15-21), coupling with peptidyI-D-amino-acid hydrolase. Unfortunately. using this procedure we were unable to obtain complete hydrolysis of the proteins, since this last enzyme can split peptide bonds of peptides composed of not more than five residues of D- or L-amino acids [6]. Therefore. it seemed necessary to split the protein chain to fragments by chemical means before the enzymatic completion of hydrolysis. A preliminary study was performed in order to establish the conditions of partial chemical hydrolysis in which the racemization of amino acids occurs at a
Rate of hydro/ysis o/proteins and comparative study of the racemization of the amino acids Fig. 2 shows a typical profile of the sequential procedure for complete hydrolysis of proteins. The results are the mean values of four hydrolyses of a crude homo-
iI" ?
E
~
0/
!,
-
--J
3
~3o
Io
'"
'
lu
3o
q 0 2 2 C~ ÷
-,','-C
!
50
70
HyClPolysts t e r n l ~ r a t u r e
90
I~0 " c
4
,
J
8
*
m
=
,
12
t
-"
~s dG),s
Hydro;ys,5 at 37 "C
Fig. I. 'Rate of racemiZatKm of anuno aods in 6 M and 10 M HCI at vanous t i m ~ and temperatures. The sample u.~:l in this expc~mcnt ~a.~ bovine serum albumin. Pand A shows the mean value oi the rate of racemization of ~ c acid and alanin¢ after hydrolysis [or 24 h m 6 M and l0 M HC! at temperatures betwen I0 o C aad 110 o C. Panel B shows the percentage or total hydrolysis ( ) of bovine scrim3 albcum,n at 37 o C in f: M and in 10 M HC1 and the percentage of racemizauon of ~partic acid t . . . . . . ) after hydrotysts of the same protons m the same condmons.
204 I
~
step~
----.~
I
step 2
i
F--
s%ep3
I
-~ - - - - - S 8O % ¢, 6 0
~
to0,
4o
o
~c ~o
$- . . . .
~_
e- . . . .
•
O-- -- ----4- ----- -Q
oL
0
4
,3
12
0
6
I)OyS
12
Hour5
.....
• . . . .
•
~ ....
4----
0
6
0015
v_
o~
~og
~ 12.
HOURS
Fig. 2. Sequential steps of hydrolysis of proteins with very. low values of amino acid racemization. The values refer to the mean of the results obtained from the following samples of protein: bovmc ~r~nt albumin, human serum, bovine leg muscle. Octop~ vulgaris hepatopancreas and homogenate of peas. - - , total hydrolysi: of proteins; . . . . . . , racen'uzation of the anuno acids. Step 1: hydrolysis in 10 M HC1 at 3 7 ° C . Step 2: enzymatic hydrolysl., with pronas¢. Step 3: enzymatic hydrolysis by leucine aminopeptidase combined with peptid~l-o-amima-acid hydrola~.
genate of bovine leg muscle (taken as an example of a mixture of various kind of proteins). Three stpes are required to obtain total hydrolysis. For the first step, tl'.e chemical hydrolysis, conditions were chosen such as suggested by the data described above, that is. low temperature and long duration of trea.'meat by 10 M HCI. The perc¢ntage of hydrolysis reached after 12 days is about 50%, while th= percentage
of racemization was kept below 0.015% (ratio~ O/D + L) for aspartic acid and 0.01% for alanine. The second step, treatment w i ~ pron~.~e ~'~, b.'5.r,~ :he hydrolysis of proteins from about 50% to about 80%, with respect to total hydrolysis. Finally, the third step completes hydrolys;.s by simultaneous treatment with leucine aminopeptidase and peptidyl-o-amino-acid hydrolase. In the last step we obtained total hydrolysis of pro-
TABLE i
l!,d,~,(, ; ~,f protem.~ bl 16 M HCi fi~r 1S days at 37°C and by enzymatw hydrolysis The values refer to the percent of tot,~l i13,drolysis of the amino acids with respect to that obtained after h_',drolysis in 6 M HCI at i i 0 * C for 24 h. A refers, tc the percent of each amino acid h)'drolyzed after treatment with 10 M HCI at 3 7 ° C for 12 days. B refers to the percent of each amino acid h}dtolyzed alter the complete hydrolysis of proteins [tO M HCI at 37 ° C for 12 days + enzymatic) treatment vnth ptonas¢ and with leu~ane anunopeptiaas¢ together w~th peptidyl-t>-amino-acid hydrolasc. -,,n,, acids
Asp t'hr Ser Glu Pro Gly Aia Va] Cy~ Met I le Leu Tyr Phe Lys !-|i ~ Arg
Bo~,me serum albumin
Human serum
Bovine mu.~le
Hcpatopancreas of
Homogenate
of leg
Octopus t'ulgarls
of pe~s
A
B
A
B
A
B
A
B
A
B
38 39 43 4~ 60 5g 56 41 4g 59 52 54 60 52 59 55 67
100 101 t~ 98 99 ll.X) 101 99 100 101 9g g9 I00 100 10t) I01 98
40 3b 47 46 65 65 46 38 50 61 50 56 73 4q 60 50 70
I00 98 100 100 qq 101 100 gO 99 1(30
45 35 49 50 70 64 48 46 59 55 M
100 ,08 100 9g IOl 100
63 ¢~ 43 54 65 60
51 43 53 4g 68 60 43 49 55 49 41 63 57 44 50 58 61
100 9g 98 97 10(.I 99 101 10l IO3 9g g7 g8 99 100 101 100 100
3~ M 55 54 73 68 44
100
99 98 !00 99 101 i(30 100 99 1(30 98 99 100 100 100 99 9~ 101
45
100 9g 98 101 100 98 100 100
65
100
63 35 62 80 43 62 70 5.5
i01 98 58 100 100 101 97 g8
205 teins with the liberation not only of all L-amino acids. but also of all of D-amino acids if they were present in the sample in examination. In order to verify that a similar pattern was ,)brained also in other proteins, either purified or a mLtture of non-purified proteins, bo~me serum albumir,, human hemoglobin, crude homogenate of octupos hepatopancreas arm crude homogenate of peas. ~ere subjected to the hydnolysis procedure described. As is shown in Table I, column A, in the first step of hydrolysis, the percentage of each amino acid released is about 40-60% ~ t h respect to total acid hydrolysis that occurs at I I 0 * C in 6 M HCI for 24 h (Fig. 1A). After the third step of hydrolysis, all the amino acids composing the proteins under examination, were completely hydrolyzed (Table !. column B). Tal~!e !! shows the percentage of racemization of each amino acid (expressed as the ratio % D/D + LL after total hydrolysis of proteins using the present method in comparison with that obtained when the proteins were hydrolyzed in 6 M HCI at l l 0 ° C for 24 h [I.3-4]. As is evident from the values listed in the same table, using the present method, after total hydrolysis of proteins, aspartic acid r a c e m e s at a percentage that is less than 0.015%, whereas the following amino acids: glutamic acid, alanine, leucine, isoleucine, valine, proline. phenylalanine, serine, methionine and tyrosine racemize less than 0.01%. On the contrary, when the proteins are hydrolyzed at elevated temperature, the
amino acids do racemize at a much higher r~te. In fact. as reported by other investigators [1-4]. under the usual conditions of total chemical hydrolysis of proteins, the amino acids ratmmize between 1 and 9% (ratio D / D + L × 100) depending on the kind of amino acid and the protein treated (Table II. superscripts a. b and c). As for the following amino acids: arginine, cystine, threonine and lysine, since the D-enantiomers of these amino acids are not oxidized by D-aspartate oxidase or by D-amino acid oxidase, they were determined by gas chromatography according to Frank et al. [14]. In ahi~ case, since the minimum value for measuring a ratio of D/L-amino acid should not he below 0.1%, the values of racemiTation for these amino acids are expressed as below 0.I~, (Table II). We were unable to evaluate the rate of racemization of the last two amino acids listed in this table, because tryptophan is destroyed in HCI, and histidine cannot be determined with gas-chromatography techniques [14].
Hydrolysis of synthetic peptides containing D-amino acids and of human cataract lenses Up to this point we have shown that using the present method, total hydrolysis of proteins accompanied by an almost negligible racemization of amino acids can be obtained, in order to prove that the method completely releases the t>-amino acids occurring in peptides or proteins, we used several commercially available synthetic peptides containing D-amino acids
T A B L E II
Comparauve stud~ cm the percentage of raceml'_at~on o/amino actd~ under different conditions o/h~drd)*'ts T h e ~alue~ refer to the percent of racemtzation e~tpres~ed as the r a t i o D / D + t X 100.
Asp Giu Ala Leu lie Val Phe Pro Ser Met Tyr Arg Cys: Thr Lys
a-Lactalhuman J
B-lactal bumnn ~
l]k~vine a plasma albumin
Insuhn"
5.3 1.9 1.2 3.7 37 2.6 1.0
4.9 3.4 1.4
72 3.6 2.. 3.3 4.9 1.1 2.4
5.6 9.~ 1.4 1.9
2.2 I 2 0.9 3.5
5.4
5.1 1.3
1.2
His Trp • b ¢ d
Hychrolysis Hydrolysis H~drolysas Hydrolysis
8.4 2.0
-
0.43 29 0.2 2.2 I.)
-l.l
9.0
Bradyktnm •
2.0
3.85 2.4 0.45 1.6~" -
Bovnne 0 ~,¢rum albumin
Human a hemoglobin
Hcpatopanereas a of Octopus vulgaris
Hom~gcnate a of peas
< 0.015 < 001 < 0.01 < O01 < 0.01 < 0 01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.1 <0.1 <0.l < 0.1
< 0.015 ~ 0.01 < 0.01 < 00! < G.01 < 0.0l < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.1 <0.1 < 01 < 0. I
< < < < < < < < < < < <
< 0.0A5 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 0.01 < 001 < 0.01 < 0.01 < 0.1 <0.l "-0.I < 0. I
for 24 h for 24 h for 22 h in 10 M
in 6 in 6 m 6 HC!
M HCI at l l 0 ° C ( L i a r d o n c t a l . Rcf. 4). M HCI a t l i 0 ° C ( F r a n k c-t al., Rcf. 3; D a t a e x t r a p o l a t e d froth, Fig. 2). M HC1 at l l 0 o C ( M a n n i n g a n d Moore. Rcf. 1). for 12 d a y s at 3 7 ° C followed by e n z y m a t i c hydrol.~s,, ~.p,e,,ent ~ o r k L
0.015 0.01 0.01 0.01 0.01 0.01 0.01 LI.01 0.01 0.01 (I.O~ I:.1
,.0.1 < 0.1 < 0.1 -
2O6 TABLE III Amount of t- and
P-amino
acids hberated from pepndes and protems hr en:ymatrc hrdrv(*,,t.~ fStep ." ond Step 3;
The concentration of amino acids liberated is calculated from the chromatographic analysis before and after ~reatment wilh D-amino-acid o~a~cs¢. The values given in parentheses are the values obtained after oxidas¢ treatment. Anuno acids liberated from 1 pmol of peptide
Synthetic pcptides
T~
DAla
Phe
Leu
IoAla2-Lcul ¢nkcphalin ( Tyr- DAla-Gly-Phc-Leu)
0.98 (0.96
1.03 I (0.96) (0)
0.96 (1.02)
0.98 (I.02)
[oAla:-DLcu s I enkephalinamid¢ ITyr-DAla-Gly- Phe-DLeu • N H ~)
(0.98)
0.98 (099)
0.97 (l.OI)
1.05
0.9~
[[)AIa2-DMeI ~] enkephalin
(Tyr-DAla-Gly- Phe-DMet)
1.02 0.96
! .03
(0) 0.97
(0.98) (0)
Gly
oLeu
DMcI
Tip
DPh¢
Scr
DArt
LArg
Pro
1.98 (2.02)
0.91,l (0)
1.02 (0.99)
0.96 (0.8)
1.02 (1.0)
0.98 (0.(16)
0.95
(0) 0.98 (0)
(0.991 (1.02)
LH-RH hormone
(AoctyI-DTIP-DpCI- Ph¢-DTip-Scr-Tyr-DArg-Leu-Arg-pro-DAla.NH2)
1.03 (0.95)
0.97 ( 1.02 )
1.02
(l.0)
Central nucleus of human lenses cataract Total chemical hydrolysis (6 M HClat I I 0 ° C for 24 h)
Total hydrolysis ~ith
total amino acids (~moi/1O mg of ti~u¢)
total aspartic acid (it tool/10 mg of tissue)
total amano acids ( p m o l / 1 0 mg of tissue)
total DAsp (jamol/10 mg of tissue)
total t.Asp (/~mol/10 mg of tissue1
total Asp (/Amot/10 mg of tisMlel
22.8
2.1
22.1
0.260 (I 2.3c~) ~
1.84
2.1
24.1
2.2
23.,)
0.256 (! 1.9~) .I
1.90
2.15
present method
Women between 71 and 76 years Men between 57 and 72 year~
" Refers to r trio q~ of t)/D + x. aspartic acid.
and human lens cateract, which contains a large amount of D-aspartic acid [22-23]. As peptides containing D-amino acids, we chose the following: [DAia2-1eucine] enkephalin. [oAla 2. DLeu s] enkephalinamide, [DAla:, D M e : ] enkephalin and the LH-RH-luteinizing hormones containing DTrp, DArg and oAla. 1 mg of each of these compounds was dissolved in 1 mi Tris-HCl buffer (pH 7.5) containing 5 mM CaCI,~ and subjected directly ta the two enzymatic hydrolyses (Steps 2 and 3) as described above, but using the amount of the hydrolytic enzymes in proportional quantities. The first step was omitted, since there was no need for a partial hydrolysis, the peptides being already relatively small. After hydrolysis, half the amount of each sample was brought to pH 2.2 and was subjected to amino acid analysis to determine the content of amino acids liberated. The other half was treated with 5 #1 of D-araino-acio ox:.dase and 5 pl of catalase to destroy D-an',ino acids and then analyzed as the first one i7]. The results obtained from this investigation demonstrated that the yield of the L- and D-amino acids was equal to the expected values. In fact, as is shown i,l Table I!I. in which the various pcptides containing D-amino acids after enzymatic hydrolysis using our procedure are reported, all amino acids consisting of the
peptides either in D or L form are completely liberated. From I /~mol of the pcptide DAla'--Leu-enkcphalin we obtained amounts of amino acids near to unity. The same results were obtained when the other three peptides were used (Table III). The eye lens cataracts studied had been sungic',dly removed from four women between 71-76 years of age, and from four men between 57 and 72 years of age. The two groups of four lenses were homogenized in distilled water and were subjected to the hydrolysis procedures as described in Materials and Methods. A portion of same samples were hydrolyzed in 6 M HCI for 24 h at I 1 0 ° C to determine the absolute content of aspartic acid. As is shown in Table !il, using an equal quantity of sample, after our hydrolysis procedure, we found a concentration of total amino acids and of total aspartic acid that was the same as that obtained from chemical hydrolysis (110°C for 24 h in 6 M HCI). Regarding specifically the content of D-aspartic acid in tl'~,c tissues, using our method, we found 0,260 /amo! of Daspartic acid and 1.84 pmoi of L-aspartic acid in 10 mg of the lenses from the women. This value equals 12.3% of the D form. ~ith respect to total amino acid ( D / D . L × 100). Since the sum of DAsp plus LAsp is equal to total aspartic acid found using total chemical hydrolysis
2O7 F----
step 1 - - ~
I
step 2
I
I----- step 3
'°° t
~C
A -112
6(:
O
j
/
/
/
B~s
/
0
0-- . . . .
~oO
C
O'-- - - - - O
v
,q ,
0
i
•
I
8
4
,
I
~2
C
~Gy~
6 ~..:r~
~2
6
12
b4OUrS
Fig. ~. Sequcmial steps of hydrol~'szs of the p¢oteln~, of human cataract lenses. The values refer to the mean of the results of hydrolysis nf eight hmscs. - - . total hydroi)~ts 04" proteins" . . . . . . . racemmation of the amino acids. Step h hydrolysis in 10 M HCI at 3 7 ° C . Step 2: hydro~vsas ~tath p r o t t ~ . Step 3: enzymatic h vdrolyms wtth leucme aminopeptJda.s¢ combined with pept~lyl-o-~mano-acid hydro4ase. In step 3. it~licates, hydrolysis earned out with: (A) Ict~m¢ a m m o p e p l i d a ' ~ and peptld~l-D-ammo acid hydrola.~; (B) I~tcilfl~ amino peptidas¢ only; (C) only with the peptidyl-t~ammo-a~d h~dtol&~. - . . . . . . amount of o-aspartic acid released from pCplidCs after treatment with: (A) |¢ucir~ amtnopepttdas¢ and pcptid),bI>-amlra3-acgt h~,drtdas¢. (B) with only ixq0tidyl-o--ammo-acid hydrolas¢, and (C) with only Ieucin¢ amanopept~das¢.
(6 M HCI, 24 h. I I 0 ° C ) , we conclude that the content of o-aspartic acid, with respect to total aspartic acid, is the real value. Approximately the same results were obtained on cataract lenses from the men (Table Ill). Fig. 3 represents the hydrolysis pattern of the eye lens cataracts. D-Aspartic acid released it. t.t~e first step was about 5~ of the total. It remains at 5~ during hydrolysis with pronase (step 2). The impor,ance of the action of peptidyI-D-aminoacid hydrolase clearly emerges in step 3. In fact, leucine aminopeptidase alone h)drolyzed almost the all L-armno acid (Fig- 3, curve B. unbroken line), but not o-aspartic acid (Fig. 3, curve C, broken line). PeptidyI-D-armnoacid hydrolase alone caused only a small increase of the total hydrolysis of L-amino acids (Fig. 3, C. unbrnken line), but it induced a 5-8c~ release of total o-aspartale present in the proteins (Fig. 3, curve B, broken line). Lastly, peptidyl-t>-amino-acid hydrolase assoc;.z:~ed with leucine aminopeptidase resulted in the total hydrolysis of proteins (Fig- 3, curve A, unbroken line) and total release of D-aspartic acid (Fig- 3, curve A, broken line). l~semskm D-Aspartate was found in the free form in the n e r v o ~ system of Octopus v~dgar~ Sepm officinalis and Loligo vuigar/s [7.24] and i>-.'flani~¢ in Crustacean muscle and hepatopancreas [25], and we hypothesized that this compound is present in proteins or peptides, from which it may be released by a specific peptidase. For these rmsons, we developed a new method of hydrolyzing peptides arm proteins with a very Io,~" degree of amino
acid racemization. This method is parucularly ~uitable for determination of the rate of racemization ol ar,.ino acids in peptides and proteins which can occur in vivo at very low, but still significant levels. A crucial point to obta,n total hydrolysis, is that leucine aminopeptidase and peptidyl-o-amino-acid hydrolase mus: be used together, because if a o-amino acid is present in peptide residues, the leucine aminopeptidase cannot split J',¢ peptide bonds consisting of two o-amino acids or by an L-amino acid and a o-amino acid. Therefore. when a o-amino ac;.~ i~ situat~,.~, in the N-terminal position, only the peptidyI-D-amino-acid hydrolase can cleave the bond. Once o-amino acid is released, the leucine aminopeptidase :ondnues to split the peptide until it meets the next t>-amino acid and so on.
To evaluate the percentage of racerrdzation, we tested aspartic acid. b ~ u a s ¢ it has a higher rate of racemization than other amino acids, and alanine, because it racemizes at a laedium rate a.g compared to other amino acids. Hence. the values obtained for these amino acids reflect the rate of racemization of other amino acids. We -used the enzymatic fluorimetric method instead of gas-chromatography techniques [141 or HPLC techniques [26-27], since it allows reliable determination of the percentage of racemization of amino acids as low as 0.01% (ratio D / D + L X 100). l>-Aspartic acid at high levels has bee~ found iL hur,~an tissues, such as lenses during ageing and in cataract lenses [22,23], tooth enamel [28-29], brain [30-31] and erythrocytes [32]. in all these studies to reduce the deodec of racemization, the proteins were
208
hydrolyzed for only 6 h in HCI at 100°C. However. also under these conditions, a considerable amount of racemization of amino acids took place. In fact. we found that under these hydrolysis conditions, aspartic acid racemizes at a rate between 0.8 and 1.27o (exp:essed as ratio D/L + t X 100), while alan(he racemizes between 0.2 and 0.4% depending on the type of proteins. In the studies mentioned above, aspartic acid racermzation values found in brain of young people were taken as blank values, because the natural racemization in these tissues is very low. The valu~ found in these tissues was subtracted form the values obtained from corresponding tissue from elderly individuals. However. it should be noted first that the ra:e of racemization of amino acid~ induced by chemical hydrolysis is influenced by neighbouring amino acids (Table ll), and second, there is no evidence tha~, the protein composition of the brain of a young person is identical to that of an elderly person. McFadden and Clarke [33] and Geiger and Clarke [34] have report that under some assay conditions of near-neutral pH salues, D-isoaspartate :esidues may arise from some peptides undergoing deamination. Since o-aspartate oxidase which we used does not oxidize such compounds, a small amount of D-aspartic acid may be underestimated. The present method, applied to the proteins and peptides used in this work, leads to total hydrol)sis. Since these compounds comprised pu6fied proteins as either whole animal or vegetable tissues, it is very probable that the method hydrolyzes all proteins. However, the amount of total amino acids obtained with the present method should be compared vdth that obtained using the classic method of chemical hydrolysis (6 M HCI at 110 o C for 24 h). In conclusion, the method described in this paper greatly reduces racemization of amino acids during the hydrolysis of proteins, h is suitable for the hydrolysis of peptides and proteins consisting not only of L-amino acids, but also of I>-atnino acids. It is lengthy compared t,~ no.qnal acid hydrolysis, but it is the only procedure yet available that permits the detection of small amounts of racemization of amino acids in vivo that wot.ld otherwise have been inde~.ectable. Acknowledgements We are grateful to Dr. Giovanna Nard( and Professor J. Manuel Denuc6 for critical examination of the manuscript. We also thank Mrs. Giuseppa Trovaglione of the Department of Biological Chemistry. of the University of Naples for performing amino acid analyses and Professor F. Menna and co-workecs, of Pellegrini Hospital in Naples. for providing human cataract lenses.
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