multiple carboxymethylation of histidines in bovine ribonuclease a

Eur. J. Biochem. 22 (1971) 225-234

Multiple Carboxymethylation of Histidines in Bovine Ribonuclease A

Jake BELLO and Eugene F. NOWOS~IAT

Department of Biophysics, Roswell Park Memorial Institute, Buffalo, New York

(Received May 21, 1971)

Reaction of RNAase A with bromoacetate a t pH 5.5 for 1-42 days results in multiple reactions. Alkylation of residues proceeds in the sequence : (1) N-1 of histidine-119; (2) methionine (probably methionine-30); (3) N-3 of histidine-12; (4) N-3 of histidine-105 and N-3 of l-carboxymethyl histidine-119; and (5) lysine-1. Both histidine-12 and histidine-119 of the same active site are carboxymethylated. A derivative carboxymethylated at both active site histidines is obtained in 1 day and probably some of this derivative is obtained in short reaction times. This is contrary to the conclusions of earlier investigations. Histidine-48 undergoes little or no reaction. The results are in accord with the X-ray structure of RNAase.

There are two histidines (residues 12 and 119) in the active site of RNAase. The alkylation of these in bovine pancreatic RNAase A at pH 5.5 with halo- acids has been extensively investigated [l - 81 in solution and in the crystal. An important conclusion of these investigations was that during reactions of several hours either histidine-1 19 yielded the l-carb- oxyalkyl derivative or histidine- 12 yielded the 3-carb- oxyalkyl derivative, but alkylation never occurred at both residues in the same molecule of monomeric RNAase. Carboxymethylation for 23 h resulted in the formation of minor amounts of unidentified new products [5]. In our work we have used reaction times of 1 to 77 days in order to study the possibility of more extensive reaction. During the preparation of our manuscript, Goren and Barnard [9,10] reported that a second carboxymethylation occurs at histidine-119. They reported the formation of a product in which both 1-CMhistidine and 3-CMhistidine appeared. They surmised that the 3-CMhistidine arose from reaction a t histidine-105, or histidine-48, or both, but not a t histidine-12 [9]. Fruchter and Crestfield [ll] found that in the dimer of RNAase, both histidine-12 and histidine-1 19 could be carboxymethylated in the same monomeric unit. A small amount of such product was obtained. In the dimer these histidines are in different active sites, and, presumably, independent of each other. In this communication we present evidence that RNAase A can be alkylated a t both histidines- 12 and - 119 in the same active site.

Unzlswll Abbreviations. CM, carboxymethyl ; RCM, reduced and carboxymethylated on cysteine.

Enzymes. Bovine pancreatic ribonuclease or polyribo- nucleotide : 2'-oligonucleotidotransferase (cyclizing) (EC 2.7.7.16); trypsin (EC 3.4.4.4).

MATERIALS AND METHODS Materials

Ribonuclease A, phosphate free, was purchased from the Worthington Biochemical Corp. (Freehold, New Jersey) ; Cleland's reagent (dithiothreitol) and trypsin (tosyl-L-phenylanylchloromethane derivative) from Calbiochem (Los Angeles, California) ; polylysine, (molecular weight 110000) from Pilot Chemicals (Watertown, Massachusetts) ; aceto- hydroxamic acid from Nutritional Biochemicals Corp. (Cleveland, Ohio) ; and bromoacetic acid and cyanogen bromide from Eastman Organic Chemicals (Rochester, New York). Bromoacetic acid was re- crystallized from chloroform and stored a t - 20 "C. s-Carbobenzoxy-L-lysine was bought from Schwarz- Mann (Orangeburg, New York) and the carboxylic acid resin (corresponding to Amberlite IRC-50) Bio- Rex 70, - 400 mesh, from Bio-Rad Laboratories (Richmond, California). The resin was conditioned as described by Hirs et al. [12].

METHODS

Carboxymethylation for 42 Days RNAase (500mg) was dissolved in 15ml of

0.1 M sodium acetate to which 50 mg of bromoacetic acid was added. The pH was adjusted to 5.5 with concentrated HCl. In one experiment carboxymethylation was carried out in sealed tubes under N,. The reaction was allowed to proceed for 42 days in the dark at room temperature. The protein was desalted on a 3 . 4 ~ 70 cm column of Sephadex G-25, medium grade, equilibrated in 5O/, acetic acid.

100 mg of the reaction product was dissolved in 1 ml of 0.2 M sodium phosphate, pH 6.47 & 0.02,

226 Carboxymethylation of Histidines in Ribonuclease A Em. J. Biochem.

and placed on a 0.9 x 150 cm column of Bio-Rex 70 maintained at 30 "C and equilibrated with the phosphate buffer. The flow rate was 30ml/h and 5 m l fractions were collected, following Crestfield et al. [13]. The material balance obtained from the integrated absorbance-volume curves of the chromatograms from the Bio-Rex 70 column, compared with the absorbance of diluted aliquots of the applied solutions, showed recoveries of a t least goo/,. After desalting by filtration through Sephadex G-25, fractions I and 111 were rechromatographed once, and fraction I1 twice (Fig.2B). One-third portions of fractions I and I1 were rechromatographed a t one time whereas the entire amount of fraction 111 was rechromatographed ; this corresponds to 36 mg of fraction I and 78 mg of fractions I1 and 111. Since fraction I1 showed a shoulder upon rechromatography (Fig. 2 B) it was rechromatographed on a 0.9 x 150 cm column of Bio-Rex 70, equilibrated with 0.3 M Tris-sulfate buffer pH 7.9 and eluted with the same buffer [14]. A load of 60 mg in 1 ml of buffer was applied and all other conditions were the same as those described in Fig.2A. The fraction cut taken for subsequent work is indicated in Fig.2B by vertical lines. The Bio-Rex 70-Tris buffer system does not improve resolution over the Bio- Rex 70-phosphate system, so that in all subsequent work rechromatography was done on the latter system. The recovered material after chromatography and desalting was 75mg of fraction I, 153mg of fraction 11, and 60 mg of fraction 111. The total yield of the three purified fractions was 288 mg or 58O/, of the original.

The Effect of Bromoacetate on RNAase with Time A solution of RNAase and bromoacetic acid

was prepared as above, without N, purging. Portions (2 ml) were withdrawn a t various times up to 77 days (see Fig. l ) , and immediately chromatographed on Bio-Rex 70.

For more intensive study of the product from the 24 h reaction, 500 mg of RNAase was treated with bromoacetate. A 250 mg load was chromatographed ; the material corresponding to peak I11 of Fig. 1 A was desalted on Bio-gel P2, with elution by 0.5O/, acetic acid, rechromatographed with the Bio-Rex 70 column and phosphate buffer described above, and desalted again. The final yield was 50 mg, loo/, of the original amount of RNAase A used.

Enzyme Assays These were done as described earlier [15].

Reduction of RNAase with Cleland's Reagent This was done as described earlier [8].

Tryptic Digestion of Reduced and Carboxymethylated RNAase

Digestion of the protein and the isolation of peptides was done by the method of Fruchter and Crestfield [Ill.

Edman Degradation This was done by the modified procedure of

Konigsberg and Hill [IS], except that the solution was extracted 8 times with 4 ml of benzene to remove the phenylthiohydantoin-histidyl derivative, and the aqueous phase was taken to dryness, hydrolyzed, and analyzed for its amino acid content.

CNBr Cleavage The CNBr procedure of Gross and Witkop [17]

was employed using the moditication of Steers et al. [18]. The protein, 13.7 mg (1 mole), was dissolved in I ml of 700/, formic acid containing 25mg (0.24 mmole) of CNBr. The solution was stirred for 18 h, and taken to dryness by allowing nitrogen gas to flow over the surface. The protein was placed on a 1.2 x 12 cm column of Bio-Rex (Hf), -400 mesh, equilibrated in water, and the products were resolved using a water-glacial acetic acid gradient [7]. Two major peaks emerged in the order, C-peptide (residues 1-13) and C-protein (residues 14- 124). Ahead of the C-peptide there appeared variable small peaks which were not identified.

Cleavage of C-Peptide with Trypsin To 0.6mg of C-peptide was added 0.05mg of

trypsin in 1 ml of 0.1 M NH4HC03, pH 8.0. After 14 h, the digest was dried with a stream of N,, while immersed in a boiling water bath. The residue was lyophilized for 4 8 h to eliminate NH4HC03. The residue was dissolved in 0.05 ml of water and applied to 47 x 56 cm 3 MM Whatman paper. The upper layer of butanol-glacial acetic acid-water (4: 1 :5 , v/v/v) was used for 24 h descending chromatography. The air dried paper was lightly sprayed with O.O25O/, ninhydrin in ethanol-acetic acid (3:1, vlv). Three ninhydrin-positive spots were seen. The spot closest to the origin was cut out and eluted with 1 M ammonium hydroxide. The eluate was evaporated in a stream of N,. Recovery of the peptide was 36O/, of theory, based on micromoles of alanine.

Reaction of E- Carbobenzoxy-L-lysine with Bromoacetic Acid

The lysine derivative, 25.4 mg (0.1 mmole) was treated with 28 mg (0.2 mmole) of bromoacetic acid in 4 ml of 0.2M Tris buffer pH 9.0 for 24 h. An aliquot was withdrawn, hydrolyzed, dried with a stream of N,, and subjected to amino acid analysis.

Vo1.22, No.2.1971 J. BELLO and E. F. NOWOSWIAT 227

Three derivatives appeared, the fastest, comprising 0.7O/, of the total amount of material analyzed, emerged under the same position as &,&-diCMlysine. The second derivative (63 O/,) emerged at the position of cystine and 3-CMhistidine. A third derivative accounting for 2.1°/, emerged after arginine. Free lysine accounted for the remainder. The first derivative must be &,or-diCMlysine and would be expected to emerge a t the same position as &,&-diCMlysine [19]. The second component must be or-CMlysine since Goren et al. [I91 found that or-CMlysine elutes after 3-CMhistidine, using a different system, whereas in our system this lysine derivative elutes a t the 3-CMhistidine position. We cannot account for the component emerging after arginine.

Hydroxylumine Test for Ester The procedure of Lipmann and Tuttle [20] was

used except that the protein precipitation step was omitted since any hydroxamate formed in RNAase would be attached to protein, and that all reagents were made up in 8 M urea (hydrochloric acid in 6 M urea). The final urea concentration when all reagents and substrates were present was 7.5 M. Acetohydrox- amic acid was used as the standard, and Beer’s law was found to be valid. That the reagent also works on high molecular weight substances was verified by detection of hydroxamate in commercial gelatin, as a result of cleavage of the so-called “esterlike” links, but which now appear to be Asx-Gly links [21].

RESULTS Carboxymethylations were carried out for I to

77 days, with the chromatographic results shown in Fig.1. After 1 day five products can be seen. The peaks marked 119 and 12 are I-CMHis-119-RNAase and 3-CMHis-12-RNAase7 which are the only sizable products formed in 1-3 h. There is a shoulder (peak IV) on the leading edge of peak 119, and two other peaks, I and 111. The apparent absence of peak I1 in Fig. I A may arise from inadequate resolution; or peak I may be peak 11. There is some shift of peak positions arising from changes in column characteristics. With time peak 119 diminished while peaks I-IV grew. Peak I11 grew for 14 days, then stayed nearly constant until the 35th day, by which time peaks IV and 119 had vanished. After 35 days peak I11 diminished. Peak I1 grew to its maximum in 28 days and remained nearly constant to the end of the experiment. Peak I grew through the entire 77 days. (The seeming regression of peak I in Fig. 1 H compared with Fig. 1 I , is probably an experimental vagary.) The rise and fall of the various peaks appears t o follow the progression 119 -+ IV --f I11 --f I1 -+ I. The analytical data to follow strongly suggest that each of these arises from the preceding peak.

I n Fig.2A is shown another 42 day run, with features similar to those of Fig. 1 H ; but the resolution is poorer because a larger load was used. Fractions 1-111 were rechromatographed and studied in detail. There was no enzymic activity in any of the fractions. The material from each peak was cleaved with CNBr or with trypsin to obtain peptides for identification of the sites of carboxymethylation. Of the 4 histidines of RNAase, histidine-12 is in the C-peptide (residues 1 - 13, obtained by cleavage with CNBr), histidine-48 in RCM-T9 (residues 40-61) and histidines-105 and -119 in RCM-TI6 (residues 105-124). The T-designations are those of IIirs et al. [22]. The prefix RCM indicates that the peptides were obtained from protein which was reduced and carboxymethylated on cysteine. The peptides will be further identified by added roman numerals corresponding to the fractions of Fig.2. The data of the tables are for reactions carried out in air. Reac- tions in N, will be mentioned where appropriate, but will not always be mentioned when the difference in amino acid content was no greater than 0.1 residue.

For Fraction I , the amino acid analyses of the whole protein and the relevant peptides are shown in Tables I and 2. Fraction I shows the loss of 1 methionine, 2.4 histidines and 1.1 lysine (0.7 in Nz). The C-peptide from this material shows the loss of 0.9 histidine, and the presence of 1.47 residues of 3-CMhistidine. The excess of 0.47 probably arises from oc-CMlysine, (from lysine 1) which appears a t the same position as 3-CMhistidine (see Methods). The total lysine accounted for in C-peptide is 1.63 (air) and 1.84 (N2) out of 2. The high value for glutamk acid arises from homoserine (from methionine).

Further identification of the site of modification of lysine was made by tryptic digestion of the C-peptide (Nz). The digest was chromatographed on paper to give 3 ninhydrin-positive spots, the slowest of which was shown by amino acid analysis (Table 2) to be RCM-T10, residues 1-7. From the content of or-CMlysine we conclude that the site of reaction is lysine- 1. If carboxymethylation also occurred a t lysine-7, which would prevent cleavage by trypsin a t this point, a peptide containing residues I - I0 would be formed. The amino acid analysis of Table 2 indicates that not more than 12O/, of such peptide can be present, based on phenylalanine. Thus, carboxymethylation of lysine in the C-peptide segment was probably mostly a t lysine-1. The two faster moving peptides were not investigated but probably are composed of residues 7 - I0 and 11 - 13. RCM-RNAase-I showed the presence of 0.46 residue of E-CMlysine. Since this is higher than that found in RCM-T10, it is possible that carboxymethylation of other lysines took place. No &-CMlysine was found in Fractions I1 and 111.

Fruchter and Crestfield [Ill, found that when 0.15 pmole of RCM-T16 peptide was analyzed after

228 Carboxymethylation of Histidines in Ribonuclease A Eur. J. Biochem.

Tube number

Fig. 1

4 days

I

77 days i,,., 10 x) 30 40 50

L Fraction A

1.0

0.8

S 0.6 f! 2 0.4 a

0.2

Eluate (ml)

1.2 E 4 1.0 0

c

0.8 a, c $ 0.6 :: 9 0.4

0.2

m -'

25 50 75 x)O ~. Eluate (ml)

Fig. 2

Fig. 1. Carboxymethylation for 1-77 days at pH 5.5. Chromatography was done with 66 mg loads on a 0.9 x 150 cm column of Bio-Rex 70 at 30 "C, with elution by 0.2 M sodium phosphate pH 6.47

Fig. 2. Carboxymethylatwn for 42 days, preparative chrmtography. Conditions as for Fig. 1. (A) Fractions cut as shown; (B) fractions 1-111 rechromatographed under the conditions of Fig. 1 (solid lines), and Fraction I1 again chromatographed

on the same column with 0.03 M Tris sulfate (dashed lines). For loads on column see Experimental Procedure

isolating this peptide from the IRC-50 column, large discrepancies were found between experimental and theoretical values. They corrected these values by running a blank chromatogram on the IRC-50 column, and subtracting the amino acids in the blank from those in the peptide. RCM-T1O-I contained substances which appeared a t the positions of aspartic acid and serine, which should not be present in this peptide. They were also found in the C-peptides and washings of the Bio-Rex column. The values in Table 2 are corrected for quantities found in column washings ; nevertheless, high values for aspartic acid and serine are still apparent. The origin of these unexpected substances is not known. However, since Table 2 gives all of the amino acids found, and since no other tryptic peptide of RNAase can give this

analysis, and since it was obtained from C-peptide, the peptide must be RCM-T10. The product from the reaction in air was not investigated as to the identity of the modified lysine. Goren and Barnard[9] also found carboxymethylation of lysine- 1.

Peptide RCM-T9-I showed no significant loss of histidine or lysine. In peptide RCM-T16 histidine-105 is the N-terminal residue ; therefore Edman degradation can be used to shown whether or not this residue has been modified. Table 1 shows that histidine- 105 is present as histidine and 3-CMhistidine in roughly equal amounts in RCM-T16-I. Although Edman degradation was not complete, comparison of the results for histidine and 3-CMhistidine with those for 1-CMhistidine and I ,3-diCMhistidine shows a clear difTerence in response to the degradation. The

Vo1.22, N0.2.1971 J. BELLO and E. F. NOWOSWIAT 229

Table 1. Amino mid analyses for fraction I The theory values given in this table are the residues of amino acids present in unmodified RCM-RNAase or peptides derived from it. The symbol “U” is for amino acids which are unpredictable; it is used for histidine, modified histidine and CMlysine. Since lysine is unmodified in most of the peptides, we have not used “U” for lysine. Also, “U” has not been used for methionine,

for which the value for native RNAase [4] has been used

Tryp-16-1

Before Edman After Edman C-peptide Tryp-9-1 Whole

RCM-protein Amino acid

Found Theory Found Theory Found Theory Found Theory Found Theory

Aspartic acid Threonine Serine Glutamic acida Proline Glycineb Alanine Valine 8-CMcysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Arginiie Histidme 1,3-DiCMhistidine 1-CMhistidine 3-CMhistidine c Total histidine Homoserine lactone &-CMlysine

15.4

12.5 12.4

9.28

4.28 3.35

8.99 7.85 3.06 2.43 2.00 5.61 2.99 8.89 3.89 1.58 0.51 0.53 2.35 4.97

0.46

(12.0)

-

15 10 15 12

4 3

12 9 8 4 3 2 6 3

10 4

U U U U 4 0

U

0.11 0 0.89 1 0.13 0 3.53 3

0 0

(3.00) 3 0 0 0 0 0 0

0.89 1 1.09 2 0.87 1 0.09 u

U - U

1.47 U 1.56 1 0.13 Id 0.07 U

- -

- - - - - -

-

2.15 2 0.69 1 1.82 2 2.87 3 1.33 1 0.03 0

4.43 4 1.85 2

0 0

0.99 1 0

0.99 1 2.09 2

0 0.96 U - U

U U

0.96 1 0 U

(2.00) 2

- -

-

-

- -

- -

1.68 2 0.10 0 0.87 1 1.08 1 1.38 2 1.04 1

3.32 4 0.56 1

0 0.88 2

0 0.74 1 0.98 1

0 0.07 0 0.42 U 0.40 U 0.51 U 0.56 U 1.89 2

0 0

(2.00) 2

-

-

-

- -

2.17 2 0

1.04 1 1.19 1 1.51 2 1.05 1

(2.00) 2 3.22 4 0.75 1

0 0.97 2

0 0.89 1 0.93 1

0 0

0.19 u 0.47 U 0.47 U 0.04 U 1.17 1

0 0

-

-

-

- -

- -

Homoserine emerges a t the same position as glutamic acid. D S-Carboxymethylhomocysteine (from S-CMmethionine) emerges at the same position as glycine. C 3-CMhistidine and a-CiWysine emerge at the same position. d The theoretical value for homoserine lactone includes homoserine (emerging with glutamic acid).

Table 2. Amino acid analysis of RCM-TlO-I

Amino acid Found Theory for Theory for RCM-T10 residues 1-10

- - Lysine 1.22

a,a-DiCM-lysinea 0.17 - -

Total lysine 1.97 2 2 Aspartic acid 0.37 0 0

- - a-CM-lysine 0.55

&-CM-lysine 0.03

Threonine 1.02 1 1 Serine 0.63 0 0 Glutamic acid 1.16 1 2 Glycine 0.16 0 0

3 3 0 1

Alanine (3.00) Plienylalanine 0.12 Aginine 0.05 0 1

- -

or Ee-diCM-lysinea

8 a,a-DiCM-lysine and e,&-diCMlysine emerge at the same position.

latter two amino acids must be at position 119. Histidine-119 is present as 1-CMhistidine and 1,3-di- CM-histidine in approximately equal amounts. Total N-terminal histidine before Edman degradation is 0.98 (0.84 in N,, theory 1.0). Total histidine in RCM-T16-I is 1.9 (theory 2.0). Thus, in fraction I , carboxymethylation occurs a t lysine-1, a t an un-

identified methionine and a t histidines-12, -105 and -119. The total unmodified histidine of the 3 peptides is 1.47 (1 5 3 in N,), compared with I .58 (1.56 in N,) in the total protein.

Total histidine for RCM-RNAase-I was 4.97 in air and 4.84 in N,. But RCM-TlO-I(N,) contained 0.55 residue of a-CMlysine and 0.17 residue of a,a-, or .s,&-diCMlysine (which are eluted a t the same position as 1,3-diCMhistidine). Subtraction of these from the total histidine of RCM-RNAase-I(N,) gives 4.12 residues for total histidine.

Fraction I1 (Table 3) showed the loss of 0.6 methionine and 2.4 histidines. The C-peptide showed loss of 0.9 histidine and the presence of 1.1 residue of 3-CMhistidine. The C-peptide also showed significant amounts of aspartic acid and serine. RCM- T9-I1 showed the apparent loss of 0.17 histidine, but since no CM-histidine was found, probably there was no significant carboxymethylation. RCM-T16-I1 showed that histidine- 105 was present as histidine and 3-CMhistidine. Histidine-119 was present as 1,3-&CMhistidine and 3-CMhistidine. The total un- mod5ed histidine of the 3 peptides was 1.4 compared with 1.5-1.6 in the intact protein. The total 3-CM- histidine of the peptides was 1.7 or 1.5 depending


Table 3. Amino acid analyses of fraction II For details see legend of Table 1

~~

Whole C-peptide Tryp-9-11 Tm-16-11

Before Edman After Edman RCM-protein Amino acid

Found Theory Found Theory Found Theory Found Theory Found Theory

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine 8-CMcysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Arginine Histidine 1,3-DiCMhistidine 1 -CMhistidine 3-CMhistidine Total histidine Homoserine lactone e-CMlvsine

15.8

13.3 12.8

9.80

4.05 3.33

9.04 8.00 3.36 2.35 2.05 6.10 3.13 10.1 4.39 1.62 0.71 0.36 1.71 4.40

(12.0)

- -

15 10 15 12 4 3

12 9 8 4 3 2 6 3

10 4

U U U U 4 0 U

0.37 0 1.11 1 0.44 0 3.31 3 - 0

0.08 0

0.08 0 0 0 0 0

0.09 0 0.93 1 1.87 2 0.94 1 0.08 U - U

U 1.11 u 1.19 1 0.21 1

U

(3.00) 3

- - - -

-

-

2.23 2 0.88 1 1.88 2 3.03 3 1.15 1

0

4.20 4 1.95 2

0 0

0.95 1 0

0.95 1 1.94 2

0 0.83 U

U U U

0.83 1 0 U

- (2.00) 2

- - -

-

- - - - -

2.36 2 0

1.10 1 1.29 1 1.80 2 1.32 1

4.13 4 0.47 1

0 1.21 2

0 0.94 1 1.10 1 0.12 0

0 0.47 U 0.67 U 0.34 u 0.63 U 2.11 2

0 0

-

(2.00) 2

-

-

-

- -

2.00 2 0

1.00 1 1.43 1 1.67 2 1.44 1

3.58 4 0.61 1

0 0.86 2

0 0.86 1 0.86 1 0.14 0

0 0.14 U 0.61 U 0.41 U 0.20 u 1.36 1

0 0

-

(2.00) 2

-

-

-

- -

Table 4. Amino acid analyses of fract;On 111 For details see legend of Table 1

Amino acid

Tryp-9-111 Tryp-16-111 C-peptide Whole

RCM-protein Before Edman After Edman

Found Theory Found Theory Found Theory Found Theory @. Found Theory

Aspartic acid Threonine Serine Glutamic acid Proline Glycine Alanine Valine S-CMc ysteine Methionine Isoleucine Leucine Tyrosine Phenylalanine Lysine Arginine Histidine 1,3-DiCMhistidine 1-CMhistidine 3-CMhistidine Total histidine Homoserine lactone

15.4

13.7 12.4 4.0 3.15

8.85 9.28 2.97 2.09 1.99 6.01 2.94

3.90 1.86

0.88 1.35 4.06

9.68

(12.0)

10.3

-

- e-CMlysine -

15 0.51 10 1.13 15 0.52 12 3.48 4 3 0.14

9 0.13 8 4 3 0.09 2 6 0.15 3 0.90

10 2.10 4 0.96

0.10 U U U U 1.19 4 1.29 0 0.23 1J -

-

12 (3.00) - - -

- -

0 2.17 1 1.11 0 1.84 3 3.05 0 1.03 0 0.05

0 3.60 0 2.00 0 0 0 0.95 0 1 1.05 2 2.00 1 U 0.92 U - U - U 1 0.92 1 U -

3 (2.oo)

- -

-

-

-

-

2 2.03 1 0.09 2 0.91 3 1.18 1 1.85 0 0.99

4 3.88 2 0.74 0 0 1.14 1 0.05 0 0.86 1 1 .oo 2 0.22 0

0.86 U U

1.00 U U 1 1.86 0 1J -

2 (2.00)

-

- - - -

2 1.93 0 0.07 1 0.91 1 1.17 2 2.37 1 1.02 2 (2.00) 4 3.50 1 0.66 0 0 1.14 0 0.12 1 0.78 1 1.00 0 0.15 0 U 0.22 U - U 0.91 U - 2 1.06 0 0 -

-

-

-

2 0 1 1 2 1 2 4 1 0 2 0 1 1 0 0 0 U U U 1 0 0

on whether we take the pre-Edman value or the 3-CMhistidine removed by Edman degradation. The total histidine of RCM-RNAase-I1 was loo/, higher than theory for the air product. For the N, product

the total histidine was close to theory a t 4.14, and the 3-CMhistidine content was 1.62 in the whole protein and 1.50 in the sum of the peptides. These differences are within the experimental error.

Vol.22,No.2,1971 J. BELLO and E. F. NOWOSWIAT 231

Fraction I11 (Table 4) showed the loss of 1 methionine and 2.1 histidines. The C-peptide showed the loss of 0.9 histidine and presence of 1.2 residues of 3-CMhistidine. RCM-T9-I11 showed the loss of less than 0.2 histidine, but no CMhistidine. RCM- Tl6-I11 showed histidine-105 present only as the unmodified amino acid, and histidine-119 only as 1-CMhistidine. (In N,, about 0.1 residue of 3-CMhisti- dine-105 was present.) The total unmodified histidine of the 3 peptides was 1.9, equal to that of the total protein.

Since RCM-Tl6 contains no lysine, the amino acids emerging at the positions of 1,3&CMhistidine and 3-CMhistidine are not lysine derivatives. From the incomplete reactions at histidine-105 and histidine-119 in RCM-T16, it appears that fractions I and I1 are mixtures. Fraction I11 may be fairly homo- geneous. It is likely that fractions I and I1 have two components each, in one of which histidine-105 is joined with di-CMhistidine-119, and in the other CMhistidine-105 with CMhistidine-119, keeping the total added negative charge constant.

Goren and Barnard [9] found that some 3-CM- histidine, in excess of that accounted for by 3-CM- histidine-12-RNAase7 was formed within 4 h, and that the amount of 3-CMhistidine equalled that of 1-CMhistidine after 18 h. They did not identify the histidine modified at N-3, but surmised that it was not histidine-12. Since we have shown that histidine-12 of 1-CMhistidine-119-RNAase becomes alkylated, and since a peak appears a t the position of fraction I11 in 24 h (Fig. 1 A), we investigated the 24 h fraction 111. The results are shown in Table 5 (columns 2 and 3). It is seen that two histidines have reacted and that one of these is histidine-12 (C-peptide). Also about one methionine has been modified. Thus, fraction I11 after 1 day appears to be the same product as after 42 days. It is quite probable that the 3-CMhistidine of Goren and Barnard also was at position-12. The C-peptide contains the two amino acid residues aspartic acid (0.32) and serine (0.60) found at longer reaction time. When native RNAase or 1-CMhistidine- 119-RNAase was treated with CNBr in like manner the C-peptide contained no aspartic acid or serine. Our yield of fraction I11 after 24 h was 10°/, after all manipulations, but 17 based on the areas under the chromatographic peaks. Goren and Barnard appear to have obtained nearly complete conversion in 24 h to their diCM-RNAase. The faster reaction of Goren and Barnard may have arisen from the use of pH 6 compared with pH 5.5 in our work.

It appeared probably that peak IV represents an earlier stage of reaction than does peak I11 and is the precursor of the latter. Analysis of this peak a t 14 days is shown in Table 5, column 4. A t 14 days the quantity of the overlapping peak 119 is low (Fig.lD), and peak IV should be relatively homo- geneous, while a t longer times peak I V becomes very

Table 5. Amino acid analyses of selected fractwns obtained from time study experiments

RCM- RCM- C-peptide Protein protein,

protein from from B h Amino acid 24 h peak I11 peak IV reaction

reaction 24 h l4day in 42day peak I11 reaction reaction old bromo-

acetate

Aspartic acid 15.0 0.32 15.2 Threonine 9.50 1.00 9.38 Serine 13.2 0.60 13.0 Glutamic acid 12.4 2.90 12.1 Proline 3.69 - 4.08 G 1 y c i n e 2.92 - 3.06 Alanine 12.0 3.00 12.0 Valine 8.79 0.05 9.08 8-CMcysteine 8.05 - - Methionine 3.26 0.05 3.14 Isoleucine 2.28 - 2.36 Leucine 2.08 - 2.07 Tyrosine 5.77 0.08 5.88 Phenylalanine 2.99 0.93 2.99 Lysine 10.6 2.20 10.7

3.98 Arginine 3.90 0.94 Histidine 2.13 0.34 2.86 1,3-DiCMhistidine 0.14 - - 1 -CMhistidine 0.90 - 1.31 3-CMhistidine 0.73 0.75 - Total histidine 3.90 1.09 4.17 Homoserine - 0.47 - Homoserine lactone - - -

- 7.97 Cystine/2 -

15.0

13.0 12.3

9.40

3.34 3.06

8.96 8.27 3.82 2.43 2.00 5.81 3.01

4.08 2.91

1.30

4.44

12.0

10.3

- - - - -

small. The amino acid analysis shows that about one histidine and one methionine have reacted.

We investigated the possibility that by-products of bromoacetate might cause the effects seen. The most likely products from bromoacetate would be : (a) glycolate and bromide ion, which would be expected to have no chemical effect, and to have no denaturing activity at 0.025 M, the highest possible concentration ; (b) bromoacetylglycolate, and higher oligomers, which might give rise to products alkylated with -CH2COOCH2-COO- groups, which on hydrolysis would be converted to carboxymethyl groups. Therefore, we incubated bromoacetate for 77 days under the carboxymethylation conditions, and used portions of this solution taken a t intervals of 7, 14, 28, 42 and 77 days to alkylate RNAase for 6 h periods. Chromatograms of these reaction products were the same as those obtained with fresh solutions of bromoacetate, including the complete disappear- ance of native RNAase. An amino acid analysis (Table 5, column 5) was made of peak 119 of the 6 h reaction mixture made with 42 day old bromoacetate. Analysis showed the loss of 1.1 histidine and the appearance of 1.3 residue of 1-CMhistidine. Thus, bromoacetate incubated for 42 days gives normal products at short times.

These control experiments would not necessarily distinguish -CH,COO- from -CH,COOCH,COO- ;


it is possible that they would give similar chromatograms. To test this possibility we treated the 42 day fraction I1 with hydroxylamine in 8 M urea to con- vert -CH2-COOCH2COO- groups to hydroxa- mates. Neither this material nor native RNAase gave a ferric hydroxamate color greater than the blank. The incubated bromoacetate solution itself gave a ferric hydroxamate color, as did aceto- hydroxamic acid. Thus, although it appears that ester forms in the reaction mixture, it does not alkylate RNAase. Or, if ester-containing groups are introduced into RNAase, they do not survive.

Incubation of RNAase under carboxymethylation conditions for 42 days (without bromoacetate) resulted in a normal chromatogram and normal amino acid analyses. Also, incubation of 1-CMhisti- dine-119-RNAase for 42 days caused no changes.

DISCUSSION It is clear that if the reaction is sufficiently

prolonged, substantially complete carboxymethylation can occur at both active site histidines. But a quite substantial proporOion of 3-CMHis-12-I-CMHis- 119-RNAase (17O/,,) is obtained in 24 h. Assuming that the same product was obtained by Goren and Barnard [9] it appears that a significant quantity appears a t quite short times under their conditions.

The f is t step in the carboxymethylation of RNAase is formation of the I-carboxymethyl derivative at histidine-119, leaving aside the smaller amount of initial reaction a t histidine-12. This is followed by reaction at a methionine (column 4, Table 4), which we assume to be methionine-30, following Goren and Barnard [lo]. By carboxymethylation of RNAase h e r , Fruchter and Crest- field [Il l obtained a mixture of two products, one modified at methionine-29 and the other at methionine-30. Our case is probably analogous to that of Goren and Barnard. The next most rapid reaction is at histidine-12, which is also in the active site. The rapid initial carboxymethylation of histidine- 119 at N-1 is believed to arise from orientation of the bromoacetate ion toward a positive charge in the active site [5,7,23]. This reaction adds a negative charge to the site, which would be expected to inhibit further alkylation. The reactivity of histidine-12 in 1-CMhistidine-119-RNAase with the anion- ic bromoacetate may arise from rotation of the 1-CM- imidazole of histidine- 119 to bring the carboxymethyl group away from the active site. This has been observed in the electron density map of crystalline I-CM- histidine- 119-RNAase complexed with 5-iodouridine- 5'-phosphate [23a]. We do not know if rotation occurs in the absence of a substrate analog. Rotation of 1-CMhistidine-119 would remove the negative charge and could restore a positively charged imidazole-119 to the active site, so that this side chain

could act as an orienting charge for carboxymethylation of histidine-12.

Next in order of reactivity are histidine-105 and N-3 of 1-CMhistidine-119. Carboxymethylation of N-3 of I-CMhistidine-119 could take place whether the imidazole is in its normal orientation (N-1 in the active site), or in its rotated orientation (N-3 in the active site) since the imidazole is at the outside and accessible. The reaction of histidine-I05 at N-3 is in accord with X-ray data, which show that this nitrogen is free, while N-1 is close to, and probably hydrogen-bonded to the terminal carboxyl of valine- 124.

If we take the reaction a t histidine-105 to repre- sent a non-facilitated reaction, we might think that the second carboxymethylation a t histidine-I19 is also non-facilitated, since these reactions appear to proceed at similar rates. This is contrary to the conclusion of Goren and Barnard [9]. Of course, the reaction at histidine-105 may be facilitated also, comparison of its rate with that of free histidine would not be conclusive, because the steric factor and the influence of the hydrogen bond with the carboxyl of valine-124 are difficult to evaluate. From the data we cannot be sure that reactions a t histidine-105 and a t N-3 of 1-CMhistidine-119 proceed a t similar rates ; we know only that in peaks I and I1 at 42 days both are about half completed. This is not sufficient to conclude that the rates are equal because fraction I is not more fully carboxymethylated at histidines-105 and -119 than is fraction 11. Perhaps there are two populations of 3-CMHis-12-1-CMHis-119- RNAase, in one of which histidine-I05 is reactive, and in the other N-3 of 1-CMhistidine-119. Goren and Barnard [9,10] found some 1,3-diCMhistidine- 119, but no CMhistidine-105. Possibly this reflects the presence of only one of the populations we have hypothesized. Goren and Barnard limited their reaction time to 24 h. Our hypothesis of two populations is speculative at present. It would be of interest to extend the carboxymethylation for a much longer time, or to subject 3-CMHis-12-l-CMHis-lI9-RNAase to a denaturation-renaturation cycle which might bring all of it to the same conformation. The X-ray structure does not show any necessary connection between histidine-1 19 and histidine-105.

Histidine-48 is the least reactive, in accord with the X-ray structure, being enclosed by numerous residues. The largest opening to the environment is too small for entry of bromoacetate, but large enough for entry of hydrogen or hydroxide ion for titration. Roberts et al. [24] observed by nuclear magnetic resonance that in 0.2 M sodium acetate the imidazole of histidine-48 undergoes exchange between two environments, while in 0.2 M sodium chloride there is less exchange. If 0.1 M acetate (used in carboxymethylation) and 0.2 M acetate (used in nuclear magnetic resonance) are equivalent, we would con-

Vo1.22, No.2, 1971 J. BELLO and E. F. NOWOSWIAT 233

clude that neither environment involves large ex- posure of the imidazole. The sulfur atoms of methio- nines-29 and 30 are about 0.3 and 0.8 nm, respectively, from histidine-48. Nevertheless, modification in this region does not alter the conformation sufficiently to expose histidine-48. Melchior and, Fahrney [25] have found that 3 unidentified histidines of RNAase react with ethoxyformic anhydride, while the fourth is resistant. Perhaps the fourth is histidine-48. Melchior and Fahrney also suggested this possibility. With this reagent, charge effects would be less important and histidines-12, -119 and -105 may have similar reactivities.

Carboxymethylation of the dimer of RNAze yields a small proportion of RNAase in which both histidine-12 and histidine-119 of the same molecule had been carboxymethylated [Ill. But in the dimer these are parts of two active sites; i.e., histidine-119 of molecule A and histidine-12 of molecule B form a site, and vice versa. Is it possible that in our case di- mers are formed which give rise to CMhistidine-12 and CMhistidine-119 in different active sites in one monomer unit, that the half-modified dimer disso- ciates to give native RNAase and 3-CMHis-12-1- CMHis-119-RNAase, and that the native RNAase repeats the cycle to give a high degree of conversion to 3-CMHis-12-l-CMHis-l19-RNAase? No, because in the work cited 1111, the dimer active sites (as in monomer) are preferentially carboxymethylated a t histidine-119 in both active sites, so that 3-CMHis- 12-l-CMHis-119-RNAase is a minor product. Since we obtain substantially complete conversion to 3-CMHis- 12- 1 -CMHis- 1 lg-RNAase, carboxymethylation of histidine- 12 must follow carboxymethylation of histidine-119 in the same active site. Therefore, we state our conclusion in the form: both histidines of the same active site undergo carboxymethylation.

Fig.1 shows that the peak for 3-CMhistidine-12- RNAase also diminishes with time. It is not clear what the fate of this molecule is, whether it is converted to the substances of fractions 1-111 or to one or more of the small peaks and shoulders seen in Fig. 1. Since about 9001, of the first carboxymethylation is a t histidine-I19 and about loo/, a t histidine-12, it is clear that carboxymethylation a t histidine-12 can follow that a t histidine-119. But the reverse is not necessarily true. Rotation of the 3-CMimidazole of histidine- 12 to bring the carboxymethyl group out of the active site appears to be difficult because it requires displacement of other residues. Therefore, it is possible that the 3-CM group may inhibit carboxymethylation at histidine- 119 if the latter requires a positive orienting and facilitating charge within the active site. But, histidine-119 is exposed to attack from without (X-ray result) and, therefore, it is probably not necessary for bromoacetate to enter the site. Prolonged 16 Eur. J. Biochem., Vol.22

carboxymethylation of 3-CMhistidine-12-RNAase may answer this question.

Lysine-1 is very accessible in the structure as analyzed by X-ray diffraction. Indeed, this side chain cannot be seen a t all, presumably because of vibrations of large amplitude.

The first product to be formed after the initial carboxymethylation a t histidine-119 is CMMet- CMHis- 119-RNAase. This suggests the possibility that carboxymethylation of methionine is a pre- requisite for additional carboxymethylations at the active site.

Acid hydrolysis of CMmethionine for amino acid analysis results in 20-35O/, reversion to methionine [ l l , 19,26,27]. Therefore, if we had one CM methionine and three methionine residues, we should expect to see after hydrolysis 3.2 - 3.35 residues of methionine, and if we had two CMmethionine and two methionine residues we should expect to see 2.4-2.7 residues of methionine. For fractions I, I1 and 111 (Tables 1, 3 and 4) a t 42 days, we find, respectively, 3.06, 3.36 and 2.97 methionine residues, an average of 3.13. Also, for fraction I11 obtained in 24 h we find 3.26, and for fraction IV, the probable precursor of fraction 111, we find 3.14 residues of methionine. These quantities show no trend from fraction IV to fraction I , or for fraction I11 from 1 to 42 days. Therefore we may conclude that only one methionine was modified. Goren and Barnard [9] observed that CMmethionine-30-RNAase is fully active enzymically. Fruchter and Crestfield [ i l l also found that the CMmethionine-30/CMmethionine-29 mixture was enzymically active. Therefore, since we have carboxymethylated one methionine and that one is most probably methionine-30, we suggest that the conformation of the active site of I-CMKis-119-CMMet-30- RNAase is substantially the same as that of I-CM- histidine-1 19-RNAase, and that the reactivity of histidine-12 did not arise from modification of methionine-30. The X-ray structure shows that a carboxymethyl group can be placed on methionine-29 or methionine-30 without conformational change, and that unless there is a conformational change the carboxymethyl groups cannot approach the active site or histidine-48. The X-ray structure is thoroughly consistent with the observations on the enzyme activity of CMmethionine-29-RNAase and CMmethionine- 30-RNAase and, the absence of effect on histidine-48.

The reactivity of histidine-12 in 1-CMhistidine- 119-RNAase inay have some bearing on Bernfield’s [28] finding that I-CMhistidine-119-RNAase retains polymerase activity. The polymerase activity of 3-CMHis-12-1-CMHis-119-RNAase is to be investigated,

Supported by Grants GM 13485 from the National Institute of General Medical Sciences and NIH-A-3942 from the National Institutes of Health.

234 J. BELLO and E. F. NOWOSWIAT: Carboxymethylation of Histidines in Ribonuclease A Em. J. Biochem.

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J. Bello Department of Biophysics, Roswell Park Memorial Institute 666 Elm Street, Buffalo, New York 14203, U.S.A.

E. F. Nowoswiat’s present address : Research Laboratoires, Hoffmann-La Roche, Inc. Nutley, New Jersey 07110, U.S.A.

multiple carboxymethylation of histidines in bovine ribonuclease a

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