A specific micromethod for determination of acetyl residues in proteins

ASALTTI(‘AL

DIOCIIEMISTRY

A Specific

55,

l-8

(1973)

Micromethod Residues

Received

June

for Determination in Proteins1

9, 1972;

accepted

April

of Acetyl

6, 1973

Since Narita (1) reported that, the amino terminal residue of tobacco mosaic virus protein was blocked by an acctyl group, the number of feature has rapidly increased prot,eins shown to exhibit thi, i: structural (see “Discussion”). A sensitive and specific method for the dctcrmination of acetyl groups in proteins would be helpful in studying this aspect of if the protein structure and elucidatin, u its functional role. Moreover, method is accurate enough, it can bc utilized for chemical determination of the minimal molecular weight of the ~)olypcptirlc chain carrying this residue. High sensitivity of the method is very desirable since t,ht relative molecular weight of the acetyl group to the polypeptide chain is very small and large amounts of many proteins are hard to prepare in the required state of homogeneity. Chemical methods for the detcrminntion of acetate lack specificity and sensitivity. On the other hand, enzymatic methods are much more advantageous in this regard. Von Korff (2) was the first to IIS a crude enzyme preparation from rnhbit heart for the determination of microamounts of acetate. This method was refined by Stegink and Vcstling of acetyl CoA (3) and b?; Stegink (41 who utjilizctl 1)urificd preparations synthetnse, mnlic dehpdrogenasc and citrate synthct,aae for tlltt determination of acetyl groups in proteins. Alt,hough this method ip sat&factory, one of the enzymes necded is not availahlc commercially, and some of its reagents arc rather expensive. Thcsc factors stem to have limited the ut8ilization of this method to some estcnt. In t’he course of a structural study on rabbit mllecle phosphofructokinnse (PFK) . we tlcvelopcd an cnzyrnatic mc~thotl for the dctermina‘This work was supportrd by grant numhcr GB-35438 Foundation and by n grant from thr Council-on-Rewnrch University. ’ To whom all corrcspondencr should 1)~ nddrcwc~d. Copyright 411 rights

@ 1973 by Acndemic Press. Inc. of reproduction in any form rcservcxd.

from

the Kationnl at, ~,ollisi:mn

Scirnw Stxte

2

KC-0

AND

YOUNATHAN

tion of acetate in proteins. The method is based on the following AI)P

Acetate + ATP s Acet)yl phosphate + Phosphoenol pyruvate + Pynwate + ATP Pvruvnte + NADH + H+ --f Lactate + NAl)+.

reactions:

+ ADP

These reactions are catalyzed by acetate kinase, pyruvate kinase, and lactate dehydrogenase, respectively. One micromole of NADH is oxidized per micromole of acetate present in the sample. This method has the advantages of being specific, sensitive, simple and economical. Moreover, all the enzymes needed for its application are available commercially in a highly purified form. Using the method described here, we have found that PFK contains one acetyl residue per 62,OOOg of enzyme. This is probably the molecular weight of the subunit of PFK. We reported previously (5) that the amino terminus of rabbit muscle PFK is blocked. The present findings suggest that the blocking moiety might be an acetyl group. We are currently investigating the exact location of this residue. METHODS

Materials. Crystalline or highest purity preparations of enzymes were utilized in order to avoid undesirable side reactions. Acetate kinase (170 units/mg) from Escherichia coli was purchased from Boehringer Mannheim Corporation. Pyruvate kinase (350 units/mg), lactate dehydrogenase (400 units/mg), bovine serum albumin, horse heart cytochrome c, ribonuclease A, dithiothreitol, N-acetyl-L-glutamic acid, N-acetyltyrosinamide, NADH, the sodium salts of ADP and ATP, and the tricyclohexylammonium salt of phosphoenol pyruvic acid were obtained from Sigma Chemical Co. Crystalline egg albumin was a product of Pcntex Biochemical Division of 1Iiles Laboratories, Inc. Rabbit muscle PFK was prepared by the method of I,ing et al. (6). All proteins were dialyzed against 3 changes of 0.01 M potassium phosphate buffer, pH 8.0, in order to remove any possible contamination of acetate. The diethyl ether used for the extraction of acetic acid was also washed repeat’edly with 0.01 N NaOH solution and with water for the same purpose. The standard acetate solution was prepared from 2 rnbf acetic acid which was neutralized t’o pH 7.5 with NaOH solution. The 2 mM acetic acid was prepared by appropriate dilution of 0.1 M solution of this acid which was standardized by titration with NaOH solution of accurately known strength. In view of the high sensitivity of the method, special precaution should be taken to select reagents, e.g., Tris, NaOH, and KOH, which are particularly free from acetate contamination; otherwise disturbingly high

blank readings will be obtained. Durin, 0 the course of this investigation, WC encountered some such samples among the salts labeled as “Analytical Reagent” grade. Likewise: some of the enzyme prcpnrnt.ions used in t,his study ore found to contain npprcciablc nmount~ of nc,etatc. Such samples have to be dialyzed, or a blank 11:~ to 1~ ti~~termine(l accurately and subtracted from sample readings. fL?TJdid!JSiS mid Estrnction of ilcetic Ac;c$. For the hytlrolyais ant1 extraction of acet,ic acid we followed the method of Xtcgillli and \:eStling (3) T&h some modificat,ions. In the rn~e of protein solution5 an aliquot caontniniug about 0.2 ~~molc of a&ate is plnrccl in a 10 X 75 mm test’ tube, frozen ant1 lyol~l~ilizeci. To the dry rnml>lc~, 0.7 ml of 6 x HCI is added and t,he tube is ~nlrd under rcducctl prcs~urc. Hydrolp;ii; is cxrricll out at. 110°C for 24 hr. After hydrolytic. the tul~(~:: :~rc chilh~ti l)eforcS opening to avoid loss of acetic acid. l$~h saml)lc is thcsll transferred rlu:tnt,itativelp to a gins:: st,oppered tube (16 X 150 mm) with the aill of 3.3 ml of cold water. E:stract,ion of the acetic acid from tllc liyciroiy~ate is brought about by adding 2 ml of cold cthcr and carefully mixing with a “Cycle” miser. The rthcrcal lnyc~r i,: then transfcrrcd to ;t second glass stoppered test tube containin, a a chilled misturcl of 0.9 ml of 0.5 .Y KOH and 0.1 ml of 1 nx Tris-Cl buffer, pII 8.7. The acetic acid is cxtrnc+ctl into the alkaline ayucous layer by careful mising. Finally t’hc ethereal layer is discarded and tile same process is rcpcatctl 5 timrs for t.ltc quantitative recovery of the acetic, acid. The alknlinc solution containing the acetate is adjusteci to pH 7.5 wit’h IIC:l, t~ransfcrrccl to :L volumetric flask and the volume is made to 2.0 ml nit11 ~~-l-:ltcr.Half ml of the lntt.cr solution is utilizeci for the clctcrn~inntion of acctntc, ~~iitc~lt~ 1)~ t,hc* (‘IIzymatic method described below. Therefore, it is porsil)le to carry out a determination in triplicxt’e on one sample of hgtirolysatc~. Enzymatic Dete,*na,ination of ilccttrtc. X (;ilfor(i rccorcling 5pec%rophotometer is uecd for the tictcrmination of the :tmoullt, of NBDII oxidized after the react’ion reaches cquililn%mi. The r(~action mistllrc (1.0 ml’) contains t’he following final concentrations: Trie-Cl buffer, pH 7.5, 50 m,\1; KCl, 20 mu; JIgSO.,, 6 11111~; clithiotreitol, 0.5 mkr; A’I‘P, 1 rnM; phosphoenol ppruvate, 1 rn~; KADH, 0.15 mnf; 20 !J (7 unit,s‘) of pyruvate kinasc, 20 111(8 units) of la&t? d(~h;vclrogcnns~,and 0.5 ml of the sample prepared as shown abovc~.Any NADH osidatiun that might O(Wlr at this stage is neglected. Finally, tltc react,ion is initiated hp addition of 20 jr1 (3.4 units) of arct#at,c kinasc and allowed to proceed to equilibrium. The amount of NAl)I-I osidizcti i R c~airuiated from the drop in absorhnncr at 340 nm using a molar cstinrtion cocfirient of 6.22 X I()“. A blank value has to be determined and suhtrarted from the s:lmplc values. For the blank, a volume of the dialysis buffrr, equal to the yolume

4

KU0

AND

TOUNATHAN

of protein solution taken, is subjected to the hydrolysis, extraction, and assay procedure described above under identical conditions. In our hands, the blank value varied between 0.23 and 0.36 optical density units. Concent,rations of proteins in solution were determined according to the following molar extinct,ion coefficients at 280 nm and pH 8.0: ribonuclease A, 9.51 X 10”; ovalbumin, 2.85 X 104; and bovine serum albumin, 4.52 X lOa. For cytochrome c (reduced form) a molar extinction coefficient of 2.78 X 10” at 550 nm and pH 6.0 was used. For rabbit muscle PFK we (5,7) have reported earlier an extinction coefficient of 10.9 for a 170 solution in 0.1 n- NaOH at 290 nm. Considering a molecular weight of 62,000 for its subunit,” the molar extinction coefficient for a single chain of this enzyme is 6.76 X lOa under the above conditions. RESULTS

AND

DISCUSSION

When predetermined amounts of acetat’e (range: 0.02-0.10 /Lmole/ml) were added to the assay mixt*ure, the oxidation of NADH was equimolar and linear with respect to acetate concentration. Although the equilibrium of the acetate kinase reaction is in favor of the formation of ATP and acetate, t,his limitation is overconic by the more favorable equilibrium of the pyruvate kinase and the lactate dehydrogenase reactions. Thus the overall reaction is stoichiometric in the direct’ion of acetate phosphorylation and NADH oxidation if allowed to proceed to completion. The time needed to reach equilibrium ranged from 35 to 95 min depending on the activity of the enzymes used and the sample being analyzed. The standard deviation of triplicate determinations, when predetermined amounts of a&ate were utilized, varied between 0.7% and 3.8%, indicating a high degree of precision. As low as 0.02 ,lmole of acetate per milliliter can be determined accurately by this method. According to the procedure described above for protein samples, this lower limit corresponds to 0.08 bmole of acetate in a prot’ein-bound form. To establish the validity of this method, it was applied to several prcteins and other biochemical compounds containing acetyl moieties. The results are reported in Table 1. The quantities of acetyl equivalents found experimentally are in reasonable agreement with the calculated or reported values. The trend toward yielding values somewhat lower than expected is probably due to the fact that the extraction procedure with cold diethyl ether does not yield 100% recovery. In the case of acetylated proteins examined, the average recovery is 90%. Therefore, multiplying the experimentally obtained value by 1.11 brings the result “Younathan, is in preparation.

E. S., Low,

T. L.-C.

K., Igou,

D. K.,

and

Nelson,

C. 4.. manuscript

DETERMINATIOK

Determination

N-Acetyl-tglutamic acid N-Acetyl-btyrosinamide Eibonuclexse il Ovalhumin Cyt,ochrome c (Horse heart,) Bovine serum albumin Phosphofrr~ctokillase (Rabbit, muscle) n ilverage of a triplicate * Based on a molecular I<. S., Low, T. L.-C. K.,

AI‘ETATE

TABLE 1 of Acet,ate Content of Cert,ain Other Biochemical Compounds Acetyl

Materials

OF

eqliivalents/mole

Experimentally found” 0.92 0.95 0.15 3.59 0 no 0.0 0.91

* f &+_ &

Proteins

and

of material Calculated reported

0.03 0.05 0.05 0.03 0.03

+ 0.m

determination. weight of 63,000 for the subunit, Igou, 1). K., and Nelson, C. 9.,

1 I 0. 4 1

00 no no 00 00

0.0 1 .Oh

or To Recovery 92 95 90 90 91

of this enzyme (Younathan, Manrlscript in preparation).

within the espcctcd value ~‘-4c/(,, the latter figure being the standard deviat,ion csl~erimentally inherent in s~wctrol)hotometric methods and the rtncertainty in the factors utilized in the determination of solid weights of various proteins. Althollgh direct determination of acetate on t’hc protein hydrolysate, i.e., \vithoutS prior extraction, would simplify the procedure, this was found impracticable. The very high ionic strength of the protein hydrolysnte :ifter neutralization and the strong absorbance of hydrolysates from conjugated proteins, c.g., cytochrome c, render the extraction step essential for the application of the enzymatic method. A limited number of proteins (8,9) has been shown to contain pyruvnte as a prosthct~ic group. However, it is unlikely that pyruvate woulcl interfere with the acetate cletermir~ation in such case?. Pyruvir acid is probably unstable under the drastic conditions of acid hydrolysis. Moreover, in solut.ion, kcto acids arc hydratccl whereas alil)hatic acids are not; therefore. pyruvic acid probably will not bc extracted to an appreciable cstcrlt by the ether. It ahoultl be noted alho that tlic equilibrium of the reaction cnt~alyzetl by pyruvatc kinase is far toward t,he production of pyruvate especially in prwcnce of lactate clehydrogenase. Any trace of pyruvate in t’he assay mixture will merely increase the blank reading (i.e., reading prior to addition of acetate kinase) without influencing the acet’ate determination. We have rcportcd earlier (5) that reagents conm~only used to idcntif)

6

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AND

YOUNATHAN

N-terminal residues of polypeptide chains failed to react with the Xterminal amino group of PFK. The application of the method described here to this enzyme suggests that, the blocking group might be an acetyl residue. Assuming one acetyl group per chain, and using a correction factor of 1.11, a molecular weight, of 62,000 is obtained for the subunit of PFK. This figure its very close to the one W? obtained by physical methods. The isolation and characterization of the peptide carrying the acetyl group in this enzyme is currently untlcrway in our laborat,ory. The specificity of this method is derived from the narrow substrate specificity of acetate kinnse. Among 12 carboxylic acids tested, Rose et al. (10) found that the enzyme from E. coli was active only wit,11 acet.ate (relative rate = 1.0) and to a much lesser extent, with propionate (relative rate 0.1). To our knowledge, the propionyl group has not been shown to exist in any of the proteins reported in the literature. Advantageously, acetate kinase does not act on formate. Certain N-formyl proteins have been detected in micro-organisms, plant, chloroplasts and mitochondria of higher animals. N-Formyl methionine was found to be the initiator of the synthesis of polypeptide chains in these systems [for a review, see Ref. (ll)]. The number of proteins sho-cvn to be acetylated at. the amino terminus is rapidly increasing. Recently, Stegink et nl. (12) published a list, of 15 such proteins in which the exact acetylated residue was ident,ified. Moreover, these authors referred to eleven other prot.eins which were shown to be acetylated although the nature of the acetylated residue was. not established. However, the following acetylatecl proteins were not. noted by Stegink et al. (12) : cucumber virus protein (131, encephalitogenic Al protein (14) and feline hemoglobin (15,163. Evidently, acetylated proteins occur in a tvide variety of living systems, namely, plams, plant viruses and vcrtebratee. The absence of microbial acetvlated prot’eins is noteworthy. However! this might, be a reflection of lack of enough studies rather than a specific taxonomical feature. On t.he other hand, highest preponderance of acetylated proteins occurs in mammalian muscle. It is of interest also to note that 4 enzymes of the glycolgtic pathway, namely, phosphofructokinase, enolase (17)) pyruvate kinase (4,18) and lactate dehydrogcnase (19-22) have been shown to be acetylated. The functional significance of this structural characteristic remains to be investigated. Probably it plays a role in the determination of t,he protein conformat,ion and hence its biological activity. The availability of a simple, accurate and sensitive method for the detection and deter4See footnote

3.

minntion of acetyl residues feature of protein structure.

sl~ould enhance the study

of this interesting

A scnsitiw, specific. and economical method for tile detection and determination of ace@1 groul)s in llrotcins and other l)iochemical substances is tlcscribcd. It is based on the utilization of acetate kinase, pyrevate kinwe and lactate del~yclrogena~e. An amount. of acctnte as low as 0.02 pnolc ‘ml can lx detected and tlcterminated quantitatively. The application or’ this method to rnhbit, muscle 1)hosphofructokinnse revealed tmllatsthis cnzymc contuinccl one acetpl group per 62,OOOg. The latter figure is prohnbly tllc molecular weight, of the subunit. of l~liorl~l~ofrt~ctokin:~se. clwxncteristic is diScussed. Tlw l~oSk3ihle 9ignific:incc of thi:: structnral

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(195s)

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AND

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