The complete amino acid sequence of bovine liver catalase and the partial sequence of bovine erythrocyte catalase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 214, No. 1, March, pp. 397-421, 1982

The Complete Amino Acid Sequence of Bovine Liver Catalase Partial Sequence of Bovine Erythrocyte Catalase’32

and the

WALTER A. SCHROEDER, J. ROGER SHELTON, JOAN B. SHELTON, BARBARA ROBBERSON, GERALD APELL, RICHARD S. FANG, AND JOSEPH BONAVENTURA Division

of Chemistry

and Chemical Engineering, Pasadaa, califmia Received September

California 91125

Institute

of Technology,

16, 1981

The data upon which the sequence of the 506 residues in the subunit of bovine liver catalase (BLC) is based are presented in detail. A partial sequence of bovine erythrocyte catalase (BEC) which accounts for 493 residues shows complete concordance with the BLC data. On the other hand, BEC has at least 517 residues, that is, an extension beyond the C terminus of the BLC data. Although normally BLC has only 506 residues, there is evidence that, at some point in its history, it also had the C-terminal extension. It is speculated that this extension is lost in BLC either through a different processing of the molecule in liver than in erythrocytes or by partial degradation in the first stages of catabolism.

Catalase (EC 1.11.1.6, hydrogen peroxide:hydrogen peroxide oxidoreductase) is a highly active almost ubiquitous enzyme in aerobic organisms. In 1961, efforts to determine the amino acid sequence of bovine liver catalase (BLC)3 were begun in this laboratory. Some data on tryptic peptides were published in 1964 (1) and then a preliminary report on almost the entire sequence in 1969 without detailed proof of f This work was supported in part by a grant (HL02558) from the National Institutes of Health, U.S. Public Health Service. This is Contribution 6526 from the Division of Chemistry and Chemical Engineering. ’ Portions of this paper are presented at the C terminus of this paper in miniprint. These portions are the Experimental Procedures, Tables I through VIII, and the isolative methodology for bovine erythrocyte catalase (BEC). 3 Abbreviations used: AEC, aminoethylcysteine; AE-cat, aminoethylapocatalase; BBA number, number of peptide in Ref. (1); BEC, bovine erythrocyte catalase; BLC, bovine liver catalase; CNBr, cyanogen bromide; CYA, cysteic acid; HEC, human erythrocyte catalase; HSE, homoserine; HSL, homoserine lactone; LAP, leucine aminopeptidase; MetSOz, methionine sulfoxide; O-cat, oxidized apocatalase; peptides, see Nomenclature; PTH, phenylthiohydantoin; and TFA, trifluoroacetic acid.

sequence (2). Recently information about the complete sequence has been used by Rossman and collaborators (3,4) as an aid to the interpretation of the three-dimensional structure of BLC by X-ray crystallography. This paper presents in detail the data upon which the sequence of BLC is based. It should be pointed out that most of the work to be reported was done when various enzymatic hydrolyses were the mainstay of sequencing methods, when various chemical modifications or cleavage methods were only being developed, and when sequenators were merely ideas. Indeed, it was the development of the cyanogen bromide cleavage which hastened the completion of the sequence of BLC. RESULTS

Because many cleavage methods were used and the resulting peptides were examined, numerous parts of the sequence of BLC were proved more than once. However, in the proof to be presented below in order from N to C terminus, only the minimal information that was needed to determine the sequence will be presented. 397

0003-9861/82/030397-25.$02.00/O Copyright All rights

0 1982 by Academic Preu. Inc. of reproduction in my form reserved.

398

SCHROEDER

Amino Acid Composition An amino acid composition of BLC which was determined in the initial stages of the work has been published (13). It was based on a molecular weight of 250,000. The sequence data provide a molecular weight of about 58,100 per subunit. The amino acid composition as published (13) when corrected for molecular weight and number of subunits is presented in Table I where the data are compared with the numbers from the sequence. The only major discrepancy is in the half-cystine values (AEC in Table I). Table I also provides a typical analysis of one lot that was used; again generally good agreement with values from the sequence is apparent. Mention should be made of analyses for half-cystine and tryptophan. The AEC of 10 analyses from reduced aminoethylated catalase averaged 3.4 residues with a range of 2.8 to 3.8 whereas the cysteic acid from oxidized samples averaged 3.8 with a range of 3.4 to 4.4; it was concluded that four residues per subunit are present. Analyses for tryptophan with barium hydroxide (11) provided four analyses which averaged 5.2 residues with a range of 4.5 to 5.8. When methanesulfonic acid was used for hydrolysis prior to analysis for tryptophan (14), five analyses averaged 4.9 residues with a range of 4.4 to 5.5. The sequence, of course, has 6 tryptophans. Nomenclature Peptides are numbered according to their position from the N terminus and are readily identified in Fig. 1. They are labeled to reflect the method of cleavage. Thus T, C, P, Th, Pa, AA, and CB stand for tryptic, chymotryptic, peptic, thermolytic, papaic, acetic acid, and cyanogen bromide peptides, respectively. Thus, T-l is the tryptic peptide at the N terminus and T-59 the last at the C terminus. When a label has only a single cleavage designation and a number, the peptide has its source in catalase itself or some derivative. However, if such a peptide was cleaved again, double notation defines it; thus T1,2-Pa-1 is a papaic peptide from the un-

ET AL.

cleaved tryptic peptides 1 and 2 of BLC. The amino acid composition of peptides with single notation are in Tables II through VII and those of peptides with double notation in Table VIII (see miniprint supplement). Consideration of the sequence will begin with T-l. For it and all T peptides, the sequence will be given, and the proof and overlaps will follow. Numbers above appropriate residues will help to define position. Prior data on tryptic peptides (1) will not be reiterated. In Ref. (1) tryptic peptides were numbered in their order of chromatographic emergence. In order to facilitate reference to already published data, peptides will be identified both in the new and old systems with the preface “BBA” to indicate the old system; thus, T-4 (BBA-12b). The results of the Edman degradation are denoted by solid arrows that point to the right. A dashed arrow represents a somewhat less certain identification. An arrow to the left indicates the results from the occasional use of carboxypeptidase. The Sequence The entire sequence is provided in Fig. 1 where the positions of various peptides are shown. Blocking group. The N terminus is blocked (1). However, attempts by positive and negative ion mass spectrometry of small derivatized or underivatized peptides from T-1,2 have failed to identify the blocking group. The possibility (1) that it is acetyl has not been substantiated. XRay data (3, 4) do not define the blocking group. T-42 (BBA-2~): X-Ala-Asp-Asn-Arg5 10 Asp-Pro-Ala-Ser-Asp-Gln-Met-Lvs. Peptide T-l has never been detected as such because of the well-known inability of trypsin to cleave an Arg-Asp bond. The amino acid sequence of this peptide as given for BBA-2a must be amended with insertion of an aspartyl residue (residue 9) after the seryl residue. The entire sequence results from hydrolysis with papain to produce the following peptides:

399

BOVINECATALASESEQUENCES

T-l&Pa-l

1 (Ala,Asp) 5

T-1,2-Pa-2

T-1,2-Pa-3 T-1,2-Pa-4

(Ala,Asp,Asn,Arg,Asp, Pro,Ala,Ser) 10 (Ser,Asp,Gln,MetSOz,Lys) 10 Asp-Gln-MetSOe-Lys 7-I

and from a more detailed examination of acetic acid cleavage which yielded these peptides T-1,2-AA-1 T-1,2-AA-2

1 X-Ala-Asp 3 Asn-Arg -

T-1,2-AA-3

5 Asn-Arg-Asp ---

T-1,2-AA-4

6 Pro-Ala-Ser --

T-1,2-AA-5

10 Pro-Ala-Ser-Asp-Gln----7 MetSOz-Lys

Peptide T-1,2-AA-1 was hydrolyzed with carboxypeptidase A under two sets of conditions. Hydrolysis at pH 8.5 in 0.2 M Nethyl morpholine acetate released 19% aspartic acid and 4% alanine. An equivalent hydrolysis in 0.2 M ammonium acetate at pH 5.5 released 37% aspartic acid and 5% alanine. The sequence of AA-I, therefore, is X-Ala-Asp. The partial sequence of BBA-2a was based solely on the results of dilute acetic acid cleavage. The peptide BBA-AA-1 which was then thought to be a single peptide was probably an equal mixture of two peptides from residues 6-8 and 10-12. The PTH degradation showed only one N-terminal amino acid because the second peptide was N terminal in glutamine which had cyclyzed. 15 T-3: His-Trp-Lys. Although this peptide has never been isolated as a tryptic peptide, its existence is based on peptides CB-

2 (Table II) and P-l (Table III). When P1 was hydrolyzed with trypsin, peptides T-1,2, T-4, and the first eight residues of T-5,6 were isolated and identified through amino acid analysis and PTH degradation. (T-1,2, of course, gave no PTH results due to the blocked N terminus.) Because the composition of these peptides equaled that of P-l except for a residue of histidine and one of lysine, it seemed likely that an undetected tryptic peptide was here. Inasmuch as both CB-2 and CB-1,2 were isolated in pure form, the N terminus of CB2 unquestionably must be the lysyl residue of T-2. Indeed, the Edman degradation of CB-2 revealed the sequence LysHis, . . . . Hydrolysis with bariumTygxide detected one residue of tryptophan in CB-2 (Table II). Because the other tryptic peptides in CB-2 are free of tryptophan, this tryptophan must reside in T-3 at position 14. 16 The Glu-Gln-Arg. T-4 (BBA-12b): position of T-4 is established by P-l which is described above and by C-l or (Arg, Ala, i:, (Table IV).

Gln, Lys, Pro, Asp, Val, Leu) 20

T-5,6 (BBA-6a):

Ala-Ala-Gln-Lys-Pro-

30

25 Asp- Val-Leu-Thr-Thr-Gly-Gly-Gly-

35 The Lys-Pro sequence, of course, prevents the cleavage into the components. The sequence of T5,6 was established (BBA-Ga), and P-l which was discussed above places it in the polypeptide chain. 40 Asn-Pro-

Val-Gly-Asp-Lys.

T-7 (BBA-12a):

Leu-Asn-Ser-Leu-Thr-

45 Val-Gly-Pro-Arg. Because the bond between T-6 and T-7 is cleaved slowly by trypsin, T-5,6,7 could be isolated as well as T-7 itself. 50 T-8 (BBA-lib):

Gly-Pro-Leu-Leu-Val-

55 Gln - Asp - Vu1 - Vu1 - Phe - Thr - Asp - Glu -

60 Met-Ala-His-Phe-Asp-Arg.

65 As BBA-llb,

400

SCHROEDER

ET AL.

IO 20 Alo-A*p-Asn-Arg-Asp-P~o-Alo-Ser-Bsp-tln-Mcl-lo-Alo-Gln-Lys-Pro-Asp-Vol-LCu-Thr-Thr-Gly-~~C-l

P-l

90

u Ile-Phe-Phe-Ile-Arg-AspIP-6-I

11-2: /l-24_ m 160 170 Alo- Leu-Leu-Phe-Pro-Ser-Phe-Ile-His-Ser-Gln-Lys-Arg-Asn-Pro-Gln-Th~-His-Leu-Lys~sp-Pro-Asp-Mel-

pJi-

17-27

17-26

210

IT-29

b

240

m

17-33

260 250 IIe-Lys-Asn-Leu-Ser-Vol-Giu-A~-Alo-Alo-Arg-Leu-Alo-His-Glu-As~-P~o-As~-Ty~-Gly-Lev-Arg-Asp-Leu-Phe-Asn-Alo-Ile-Alo

J

Gly-Asn-Tyr-Pm-SW-Trp-Thr‘C-16

k

230

220

m

)7-28

200

190

p%z-

IS0

P-7

EBA-c-12

260 Leu-Tyr -Ile-Gin-Vol-Met-Thr-Phe-Ser-Glu c-17--l--c-l6I

FIG. 1. The amino acid sequence of bovine

290

liver

270 -Thr-

c-15-

300

catalase (BLC).

BOVINE

CATALASE

SEQUENCES

pi?-

IT-)9 360

350 340 Lc~-Alo-Phe-Asp-Pro-Scr-lsn-Mel-Pro-Pro-Gly-Ile-Glu-P~o-Ser-Pro-Asp-Lys-Mel-Leu-Gln-Gly-Afg-Leu-Phe-Alo-Tyr-P~o-Asp-Th~~ P-9 1 L-c-25$ &P-F

p-4017-41 370 His-Arg-His-A~g-Leu-Gly-Pro-Asn-Tyr-Leu-Gln-Ile-Pro-Vol-Asn-Cys-P~o-Ty~C-26

hIT-43 p-44 380 Arg-Alo-Arg-Vol-Alo-Asn-Tyr-GIn-Arg-Asp-Gly-ProP-IO --

P-l,-

I

410 400 Met-Cys-Met-Met-Asp-Asn-Gln-Gly-Gly-AIo-Pro-Asn-Tyr-Tyr-Pro-Asn-Ser-Phe-Ser-Alo-Pro-Glu-His-Gln-Pro-Se~-Alo-Leu-Glu-HisTh-6 Th-9

@FS!J

IT-W460

h 470 -Ile-Alo-Gly-His-Leu-Ly~~-Ala-Cln-Leu-Phe-Ile-Gln-LysI Ah-9 P-13

17-57 500

IT-Jb Phe-Ser-Asp-Vol-His-Pro-Glu-Tyr-

490

390

420

IT-55 IT-St

h 480 Lys-Alo-Vol-Lys-Asn-

hIT-59

I-

FIG. 1-Catinued.

only the N-terminal glycyl residue was placed in sequence, and only two valyl residues were assigned although the analysis showed 2.27 residues after 24 h of hydrolysis. The low value for valine is, of course, explained by the valyl-valyl sequence. The sequence of T-8 was established from peptides from various cleavage procedures on catalase itself because T-8 could not be isolated in sufficient yield for extensive work on the peptide itself. Thus,

45 P-2 Leu-Thr-Val-Gly-Pro-Arg-Gly------T-T-(Pro, Leu, IZu) joins T-8 to T-7. When P-2 was hydrolyzed with trypsin, the peptide P-2-T-1, GlyPro-Leu-Leu was isolated. The sezd ----7 degradation was not good, and in fact T8 could not be degraded beyond the N terminus despite the fact that the sequence

402

SCHROEDER

Gly-Pro- from other proteins was degraded successfully in this laboratory. The remainder of the internal sequence was elucidated from the following peptides (See Tables III to VI for analyses).

ET AL.

c-3

65 Asp-Arg-Glu- Arg-Ile-Pro-----70 Glu-Arg-Val- (Val,His) --80

40 (Lys,Leu,Asn,Ser,Leu,Thr,Val, 45 Gly,Pro,Arg,Gly,Pro,Leu, 50 Leu,Val,Gln)

gether by C-4.

Th-1

50 Leu-Val-Gln-Asp ---7

75 80 C-4 Ala-Lys-Gly-Ala-Gly-Ala-Phe ---Y-T

P-3

51 Val-Gln-Asp ---

AA-2

55 Val-Val-Phe-Thr ---

P-4

57 Thr-Asp-Glu --

c-2

60 Thr-Asp-Glu-MetSOa-Ala-His---IT .-------;.- _ -, Phe (BBA-C-18)

Although the Ala-Lys of C-4 might be T14 rather than the C-terminal portion of T-11, C-6 to be discussed below precludes this possibility. 93 T-13 (BBA-18b): Tyr-Ser-Lys. Peptide C-5 effectively joins T-13 to T-12 85 C-5 ----s---Glu-Val-Thr-His-

AA-l

AA-3

60 Glu-MetSOa-Ala-His-Phe.

Finally, tryptic hydrolysis of CB-2 produced a peptide of composition equal to that of T-8 but for the five residues following the methionyl residue, and tryptic hydrolysis of CB-3 yielded this peptide 61 Ala-His-Phe-Asp-Arg --------x266 T-9 (BBA-15a):

T-12 (BBA-24b):

Gly-Ala-Gly-Ala-Phe-

85

90

Gly-Tyr-Phe-GluVal-Thr-His-Asp-IleThr-Arg. T-11 and T-12 are joined to-

(Asp,fi,Thr,Arg,Tyr). Of T-13 and T-57, the only two tryptic peptides in catalase N terminal in tyrosine, only T-13 lies within the confines of CB-3. 96 T-l.4 (BBA-14b):

Ala-Lys.

100 T-15 (BBA-23b): Val-Phe-Glu-His-IleGly-Lys. Peptides T-13, T-14, and T-15 are

joined together by C-6. (CB-3-T-1).

95 C-6 Ser-Lys-Ala-(Lys,Val,Phe) ---

Glu-AT-Q.

105

70 T-10 (BBA-14~): Ile-Pro-Glu-Arg.

T-16: Arg. T-16 is joined to T-15 as well

75

as to T-17 by C-7 and Th-3.

Tryptic peptides T-8 through T-11 are joined together by these peptides:

C-7

100 105 (Glu,His,Ile,Gly,Lys,Arg)

65 Phe-Asp-Arg-Glu-Arg-Ile-Pro----770 Glu-Arg

Th-3

105 Ile-Gly-Lys-Arg-Thr-Pro -7-----z-

T-11 (BBA-22a):

Th-2

Val-Val-His-Ala-Lys.

110 T-17 (BBA-14a): Thr-Pro-Ile-AlaValArg. T-17 is the only tryptic peptide with

BOVINE

CATALASE

the N-terminal sequence Thr-Pro. Therefore, the Thr-Pro portion of Th-3 must derive from T-17. 115 T-18,19 (BBA-13~): Phe-Ser-Thr-Val120 Alu-Gl~-Glu-Ser-Gly-Ser-Ala-Asp-Thr125 Val-Arg-Asp-Pro-Arg. The proof of linkage of T-18 to T-17 resides in peptic peptide P-5,

acetic acid prevented purification by passage through Sephadex G-50. However, the contaminants did dissolve and the insoluble portion proved to be of good purity (Table VII). Hydrolysis with thermolysin for 16 h at 4O”C, and chromatography4 on a 0.6 X 60-cm column of Aminex 50-X4 spherical resin yielded the following peptides: T-21-Th-1

110 P-5 Val-Arg-Phe. -However, peptides T-17, T-18, and T-46 are C terminal in Val-Arg. Because T-1819 is a double tryptic peptide, T-18 is excluded as a source of the Val-Arg portion of P-5. Likewise, P-12 (residues 442-445) eliminates T-46. Consequently, the Val-Arg of P-5 derives from T-17. On the other hand, T-18, T-21, T-30, and T-46 are N terminal in phenylalanine. Peptides C-9, C-13, and AA-8 remove all but T-18 from consideration. 130 T-20 (BBA-17b): Gly-Phe-Ala- Val-Lys. Chymotryptic peptide C-8 which was described in detail as BBA-C-5 provides the link between T-19 and T-20. 135 140 T-21: Phe-Tyr-Thr-Glu-Asp-Gig-Asn145 Trp-Asp-kVal-Gig-Asn-Asn-Thr150 155 Pro-Ile-Phe-Phe-Ile-Arg. This peptide was not reported in Ref. (1) because it is normally found in the insoluble tryptic core. In a preliminary report (2), only four instead of five aspartyl residues were attributed to this section. Furthermore, IleArg which as BBA-21a was considered a tryptic peptide derives, in fact, from the nonspecific cleavage of T-21 during long tryptic hydrolyses. For the isolation of T-21, 1.3 g of AEcat were hydrolyzed with trypsin for 4 h. As the pH was lowered, precipitates were removed at pH values 6.5, 5.3, and 4.3. The pH 4.3 precipitate amounted to 25 mg and amino acid analysis showed that it was primarily T-21. Insolubility of T-21 in 25%

403

SEQUENCES

T-21-Th-2

T-21-Th-3

T-21-Th-4

135 140 (Phe,Tyr,Thr,Glu,Asp,Gly, Asn,Trp,Asp) 140 Tyr-Thr-Glu-Asp-Gly-Asn/T///T Trp-Asp 140 (Thr,Glu,Asp,Gly,Asn, Tw Asp) 145 Leu-Val-Gly-Asnjr--150 Asn-(Thr,Pro) + Phe -

T-21-Th-5

145 150 (Val,Gly,Asn,Asn,Thr,Pro)

T-21-Th-6

151 Ile -Phe

T-21-Th-7

152 Phe-Phe

T-21-Th-8

155 Ile-Arg

The Edma; degradation of T-21-Th-2 places aspartic acid at residue 139 and asparagine at 141. Aspartic acid is placed at 143 because this peptide, T-21-Th-1, and T-21-Th-3, all showed less than two equivalents of ammonia on analysis. 145 Because AA-4 is Leu-Val-Gly, this peptide presumably is%nGched between 4 For the separation, the first gradient was as follows: Mixer held 166 ml of 0.1 M pH 3.1 buffer and the reservoir 83 ml of 1 M pH 5.0 buffer. The second gradient was 42 ml of 1 M pH 5.0 buffer (mixer) and 83 ml of 2 M pH 5.0 buffer. Compositions are based on those in (9) and differ in molarity.

404

SCHROEDER

residues of aspartic acid or asparagine. Consequently, T-21-Th-4 is placed after T21-Th-3 in the sequence. The placing of threonine and proline in the sequence Thr-Pro at residues 149 and 150 is based on two pieces of evidence. When T-21-Th-5 was hydrolyzed with LAP and analyzed directly, valine, glycine, and asparagine were found in the molar ratio of 1:1:1.5. Consequently, the peptide ends in Thr-Pro. Had it ended in Pro-Thr, LAP would have released only one molar equivalent of asparagine. Likewise, Th-4, that 145 150 is Leu-Val-Gly-Asn-Asn-Thr-Pro from ---;r AE-cat had some indication of threonine at the sixth degradation. Peptide T-21-Th-7 showed only phenylalanine after acid hydrolysis and no phenylalanine when analyzed without acid hydrolysis. Therefore, a Phe-Phe sequence is derived because T-21 has three phenylalanines. The C-terminal phenylalanine of T-21-Th-6 and the N-terminal phenylalanine of T-21-Th-7 must be common to give rise to an Be-Phe-Phe sequence. Because Ile-Arg must represent the C-terminal portion of T-21, the Be-Phe-Phe fragment must lie between peptides T-21-Th-4 and T-21-Th-8. Although T-21-Th-4 contained free phenylalanine as a contaminant, the sequence of T-21-Th-4 was verified when this peptide was isolated from a thermolytic hydrolysate of AE-cat (see Th-4). Another peptide that relates to T-21 is C-9, that is 135 Ala-Val-Lys-Phe-Tyr --T/ which provides the necessary overlap between T-20 and T-21. The Ala-Val-Lys portion of C-9 must come from T-20 and not T-54 because T-20 was found in a tryptic hydrolysate of CB-3. T-22 (BBA-16a): Asp-Ala-Leu-Leu160 165 Phe-Pro-Ser-Phe-Ile-His-Ser-Gln-Lys. T-22 is joined to T-21 by P-6 155 Ile-Arg-Asp-Ala-Leu-Leu -/--

ET AL.

169 T-23: Arg. An overlapping fragment which places an arginine between T-22 and T-24 could not be detected. However, the recovery of twice the molar yield of arginine over all other peptides from a tryptic hydrolysate of CB-3 first suggested the presence of two sequences from which trypsin would release free arginine. Isolation of peptide Th-3 (see T-16) excluded the possibility that the two arginyl residues could be adjacent at positions 105 and 106. Firm overlaps throughout the remainder of CB-3 excluded all other positions except that between T-22 and T-24. Because of the lability of Asp-Pro bonds which, in this instance, are present at residues 127-128 and 177-178, CB-3 was subjected to strong formic acid cleavage, and the cleavage products were passed through four 2.2 X 160-cm columns of Bio-Gel P100 (see Experimental Procedures). The peptide (CB-3-FA-1) that most closely resembled in amino acid composition that portion between residues 128 and 177 was purified by countercurrent distribution by 0.5 N HOAc:2-butanol:lO% dichloroacetic acid in the ratio of 10:9:1 and then by passage through two 0.9 X 160-cm columns of Sephadex G-50 in 25% acetic acid. CB-3FA-1 should contain either two or three arginyl residues depending upon whether or not T-23 actually exists. Amino acid analysis showed 3.0 residues of arginine (Table II). Hydrolysis with trypsin produced equimolar amounts of T-20, T-21, T22, and free arginine. Consequently, it is concluded that T-23 is arginine and lies between T-22 and T-24. 170 T-2.4(BBA-2Oa)):Asn-Pro-Gln-Thr-His175 Leu-Lys. T-24 was found as such in a tryptic hydrolysate of CB-3 and in combination with the four N-terminal residues of T-25. Therefore, it may be concluded that T-24 is the penultimate tryptic peptide of CB3 because all other sections of CB-3 have been accounted for. 180 T-25,26: Asp-Pro - Asp-Met - Vu1- Trp 185 190 Asp-Phe-Trp-Ser-Lm-Arg-Pro-Glu-Ser-

BOVINE

CATALASE

200 195 Leu-His-GlnVal-Ser-Phe-Leu-Phe-SerAsp-Arg. This double tryptic peptide nor-

mally is present in the insoluble part of a tryptic digest. Long tryptic action (24 h) on catalase liberates a peptide which contains the five C-terminal amino acids of T-25,26. It was in low yield and is described under BBA-18d. Much of the sequence of T-25,26 has been determined with CB-4 (residues 181-211). The Edman degradation of CB-4 gave these results: 185 CB-4 Val-Trp-AspPhe-Trp-Ser-Leu---T-r-Arg-Pro- -.~’ ---_.- . . . . Peptic and chymotryptic hydrolyses of CB4 yielded useful products. 190 185 CB-4-P-1 Trp-Ser-Leu-Arg-ProGluT--T--Ser-Leu 195 CB-4-P-2 (His,GlnVal,Ser,Phe,Leu) 195 CB-4-P-3 Val-(Ser,Phe,Leu) 200 CB-4-P-4 Phe-Ser-AspArg-Gly-Ile-T---7 205 (Pro,Asp,Gly,His,Arg, 210 His,HSL) 190 CB-4-C-1 Ser-Leu-Arg-Pro-Glu-Ser7--F----Leu 195 CB-4-C-2 His-Gln-Val-Ser-Phe CB-4-C-2 corresponds to BBA-C-19 (C-10 of Fig. 1). The above-mentioned peptide BBA18d which is Leu-Phe-Ser-Asp-Arg completes the sequence of T-26. That T-25 had the sequence Asp-ProAsp-Met at its N terminus and that CB4 followed CB-3 proved to be difficult tasks. They were achieved only after T24,25,26 was isolated. This was done by isolating and then removing the heme

SEQUENCES

405

from the insoluble material of a tryptic digest. The resultant product was dissolved in 50% acetic acid to which n-propanol was added to equal 20% by volume. By chromatography on a 0.9 X loo-cm column of Dowex 50-X2, peptides T-24,25,26 as well as T-25,26 and T-34 (Table VII) were isolated. Developers for this chromatogram used the pH 3.1 and pH 5.0 buffers (9) and n-propanol in the ratio of 8:2, respectively. T-24,25,26 has the residues of T-24, T-26, and the C terminal eight residues of T-25 as already determined above plus the expected two aspartic acid and one each of proline and methionine. Cleavage of T-24,25,26 was done with CNBr and T-24,25,26-CB-1 was isolated: T-24 T-24,25, ( Asn ,Pro,Gln,Thr,His, 26-CB-1 175 T-25 180 Leu,Lys, Asp ,Pro,Asp,HSL). Indeed, this same peptide had been isolated from a tryptic digest of CB-3 (see T24). Because the ammonia content from the analysis was 2.5 residues and T-24 contains both asparagine and glutamine, there cannot be amide groups in the T-25 fragment. The partial sequence then must be either Asp-Asp-Pro-HSL or Asp-ProAsp-HSL but not Pro-Asp-Asp-HSL because of tryptic specificity. The sequence was proved by the following experiment. When portions of T-24,25,26-CB-1 were heated in 0.25 N HOAc at 110°C for 8 and 16 h, respectively, and analyzed directly, free aspartic acid and proline were identified. From the specificity of cleavage by dilute acetic acid, the second sequence must be the correct one; no free proline would have resulted if the first were correct. The recoveries were less than expected; both the aspartic acid and the proline were about 0.6 residue low. This may be due to diketopiperazine formation. These data then complete the sequence of T-25,26 and show the joining of T-24 to T25,26. 205 T-27 (BBA-17~): Gly-Ile-Pro-Asp-GlyHis-Arg. AA-5 and Th-5 provide the overlap to T-26.

406 AA-5 Th-5

SCHROEDER

205 Arg-Gly-(Ile,Pro) -7 200 Leu-Phe-Ser-Asp-Arg-Gly-Ile----7 / --7 210 205 (Pro,Asp,Gly,His,Arg,His) 215

210 T-28: His-Met-Asp-Gly-Tyr-Gly-Ser-

220 T-28 has not been isolated in its entirety from any tryptic hydrolysate of BLC, but was found in impure form from a tryptic hydrolysate of bovine erythrocyte catalase (BEC) in which normally insoluble material had been dissolved prior to chromatography by reducing the pH to 2. However, it is from the pieces that result from CNBr cleavage that the structure of T-28 from BLC has been built. Thus, from a tryptic hydrolysate of CB-4, the peptide His-HSL (which must be the C terminus of CB-4) was isolated. Likewise, a tryptic hydrolysate of CB-5 yielded CB-5-T-1 His-Thr-Phe-Lys.

CB-5-T-1

215 (Asp,Gly,Tyr,Gly,Ser,His, 220 Thr,Phe,Lys)

Unfortunately, the Edman degradation failed, and when the whole CB-5 peptide was degraded, rather inconclusive results suggested a sequence of Asn-Gly-Tyr. The remaining five amino acids but for lysine then very likely correspond to C-11 (BBA215 C-16) Gly-Ser-His-Thr-Phe. The follow-7-T

ET AL.

acid. After lyophilization of the dilute acetic acid, the material was applied to new strips and again the Edman degradation was performed. The sequence G& Tyr-Gly-Ser-His was then found. J_// Because intact T-28 could not be isolated, it must be shown that CB-4 and CB5 are joined. Evidence for this linkage was obtained from material in zone 2 of Fig. 1 from (8). When zone 2 was subjected to countercurrent distribution, a peptide that closely resembles CB-4,5,6 (Table II) was isolated. The peptide is only in low yield because of incomplete CNBr cleavage. Because CB-6 definitely follows CB-5, the isolation of CB-4,5,6 indicates the sequence relationship of CB-4 and CB-5 and the existence of T-28. 225 T-29 (BBA-Sa): Leu-Val-Asn-Ala-Asp-

230 Evidence for the joining of T-29 to T-28 comes from C-12 (BBA-C-2) and from a tryptic hydrolysate of CB-5 which allows no other position for T-29 because all other tryptic peptides of CB-5 are clearly placed. 235 T-30: Phe-His-Tyr-Lys. This peptide was not reported previously. Because of its highly basic and aromatic nature, it may not elute from Dowex 50. It is unlikely that it was part of insoluble portions. When CB-5 was hydrolyzed with trypsin and the hydrolysate was chromatographed on Dowex 1, T-30 emerged first. Three steps of the Edman degradation completed its sequence. When rechromatography of this peptide was attempted on Dowex 50, the peptide was lost. C-13 (BBA-C-13) provides the overlap between T-29 and T-30: Gly-Glu-Ala-Val-Tyr-Cys-Lys.

ing peptide was isolated from a thermolytic hydrolysis of BLC. 215 235 Th-6 Met-(Asp,Gly,Tyr,Gly,Ser,His,Thr) C-13 CyS03H-Lys-Phe-His-Tyr. --/-Becausee ammonia content from the analysis was only 0.4 residue, residue 212 240 T-31 (BBA-7a): Thr-Asp-Gln-Gly-Ilemust be aspartic acid. The Edman degLys. Joining to T-30 is supplied by C-14 radation failed after the first degradation. However, the paper strips which were used and Th-7: as carriers for the peptide during the Edman degradation were eluted with 50% C-14 Lys-Thr-Asp-Gln-Gly-240 ------Y-7 acetic acid. The extract was blown dry, and (Ile,Lys,Asn,Leu) the residue was cleaved with dilute acetic

BOVINE

Th-7

CATALASE

235 (Phe,His,Tyr,Lys,Thr,Asp, 240 Gln,Gly)

245 T-32 (BBA-7’~): Am-Leu-SerVal-Glu250 Asp-Ala-Ala-Arg. C-14 described above

unites T-32 to T-31. 255 T-33 (BBA-16b): Leu-Ala-His-Glu-Asp260 Pro-Asp-Tyr-Gly-Leu-Arg. T-33 follows

Because a methionyl residue is in the peptide, CNBr peptides provided parts for sequencing. Thus the N-terminal section of T-34 (residues 263-283 which may be called T-34a) from the C terminus of CB5 was isolated from a tryptic hydrolysate of CB-5. However, T-34a maintains much of the character of T-34 and is difficult to isolate. The following peptides from AEcat belong in T-34a. 270

C-15 (Asn,Ala,Ile,Ala,Thr,Gly,Asn,Tyr, 275

Pro,Ser,Trp)

T-32 on the basis of P-7. 255 250 P-7 Ala-Ala-Arg-Leu-Ala-His-Glu-T----T 260 Asp-(Pro,Asp,Tyr,Gly,Leu) 265 T-34 Asp-Leu-Phe-Asn-Ala-Ile-Ala275 270 Thr - Gl y - Asn - Tyr - Pro - Ser - Trp - Thr 285 280 Leu-Tyr- Ile -Gin- Val-Met-Thr-Phe-Ser295 290 Glu-Ala-GluIle -Phe-Pro-Phe-Asn-Pro 300 -Phe-Asp-Leu-Thr-Lys. This peptide is

part of the insoluble material from tryptic digestion and has not been described previously. It was isolated in low yield but good purity (Table VII) by chromatographing tryptic material insoluble at pH 6.5 on a Dowex 50-X2 column with 20% propanol in the buffer (see procedure under T-25,26). A better yield of less pure material came from passage of tryptic material insoluble at pH 6.5 through four 2.2 X 150-cm Sephadex G-50 columns in series. Edman degradation of the latter material gave the sequence of nine residues: 265

Asp-Leu-Phe-Asn-Ala-Ile--T -7270

Ala-Thr-Gly-. ---s-T-

.. .

Chymotryptic peptide BBA-C-12 provides the connection of T-34 to T-33.

407

SEQUENCES

275

C-16 Gly-Asn-Tyr-Pro-Ser-Trp --7----z-277

C-17 Thr-Leu-Tyr --280

C-18

Ile -Gin-Val-Met ----

C-18 is part of BBA-C-17 and comprises the C-terminal portion of T-34a. The single glycyl residue in T-34 was detected in the ninth residue of T-34 (above) and is the N terminus of C-16. C-17, therefore, falls between C-16 and C-18. T-34b (residues 284-300, the remainder of T-34), on the other hand, was isolated from a tryptic hydrolysate of CB-6 and provided the sequence Thr-PheSer- . . . . A chymotryptic hydrGa=f the insoluble tryptic material of catalase yielded the following two peptides: 290 C-19 -Ser-Glu-Ala-GluIle -Phe-Pro---:’ ----sT---zr 295 Phe-(Asn,Pro,Phe) 300

C-21 Asp-Leu-Thr-Lys -T----TFrom a chymotryptic hydrolysate of AEcat C-20 was isolated 295 C-20 Asn-Pro-Phe ------zand from a chymotryptic hydrolysate of CB-6, CB-6-C-1

408

SCHROEDER

285

CB-6-C-1

(Thr,Phe,Ser,Glu,Ala,Glu, 295 Phe,Pro,Phe,Asn,Pro, Phe,Asp,Leu)

ET AL.

290

Ile,

325

Phe-Ala-Glu-Val-Glu-Gln-Leu /T--T-

P-9

Ala-Phe-Asp-Pro-Ser-Asn---r-r

335

These complete the sequence of T-34b and of, T-34.

340

MetSOz-Pro-(Pro,Gly,Ile,Glu, --

305 T-35(BBA-24a):

Val-Trp-Pro-His-Gly-

345

Pro,Ser,Pro,Asp,Lys,MetSOZ, Leu,Gln,Gly,Arg,Leu)

310 Asp-Tyr-Pro-Leu-

330

C-24

Ile -Pro- Val-Gly-Lys.

This peptide, previously identified as BBA24a, was successfully degraded through eight steps. The remainder of the sequence was derived from P-8.

340

345

CB-7 Pro-Pro-Gly-Ile-Glu-Pro-Ser-/---Y----T Pro-Asp-Lys-HSL

310 315 P-8 Pro-Leu- Ile -Pro-Val-Gly-Lys-Leu 7---s----

A di1utzc-E acid hydrolysis of P-9 liberated peptide P-g-AA-1 or Lys-

and linkage to T-34 came from C-22.

MetSOz-Leu-Gln-Gly-Arg-Leu which, in ---fact, provides the union with T-38. C-23 as well as the double tryptic peptide, T-36,37 (BBA-7d), supply proof of chain continuity.

300 C-22 Asp-Leu-(Thr,Lys,Val,Trp,Pro, --%5 His,Gly,Asp,Tyr,Pro,Leu) 315 T-36 (BBA-17a): Leu- Val-Leu-Am-Arg.

An excellent overlap for T-35 and T-36 was obtained from a dilute acetic acid hydrolysis of apocatalase: AA-6

Tyr-Pro-Leu--7 310 315 ( Ile ,Pro,Val,Gly,Lys,Leu, Val,Leu)

320 T-37 (BBA-la): Asn-Pro-Val-Asn-Tyr325 330 Phe-Ala-Glu-Val-Glu-Gln-Leu-Ala-Phe335 340 Asp-Pro-Ser-Asn-Met-Pro-Pro-Gly-Ile345 Glu-Pro-Ser-Pro-Asp-Lys. When this

peptide was partially characterized as BBA-la, the Edman degradation failed on the first step and it was incorrectly assumed that a glutamine residue at the N terminus had cyclyzed. Combination of data from the following peptides provided the complete sequence of T-37. 320

C-23

Asn-Arg-Asn-Pro-Val-Asn-Tyr /I----

-

350

T-38 (BBA-19a):

350 Met-Leu-Gln-Gly-Arg.

This peptide is adjacent to T-37 as already described above. 355 T-39 (BBA-23~): Leu-Phe-Ala-Tyr-Pro360 AA-7 from a dilute Asp-Thr-His-Arg.

acetic acid hydrolysis of apocatalase revealed the position of T-39 in the polypeptide chain. 350

AA-7

Lys-MetS02-Leu-Gln-Gly-Arg----z-----Y=---355

Leu-Phe-Ala-(Tyr,Pro) --T-40 (BBA-25a):

363 His-Arg.

On the basis

of C-25, 360 C-25

Ala-Tyr-Pro-Asp-Thr-His_r_--Arg-His

T-39 is followed by a tryptic peptide that is N terminal in histidine. Although three tryptic peptides in catalase are N terminal in histidine, only T-40 exists within the confines of CB-8 and therefore must be joined to T-39.

BOVINE

CATALASE

365 T-41 (BBA-4b): Leu-Gl y-Pro-Asn- Tyr370 375 Leu-Gln-Ile-ProVal-Asn-Cys-Pro-TyrArg. The Edman procedure established the

sequence of the first six residues of this peptide and the remaining residues were ordered from P-10: 375 370 P-10 Leu-Gln-Ile-Pro-Val-Asn----7--s380 CyS03H-Pro-Tyr-Arg-Ala--saJZn) -

365 C-26 Arg-Leu-Gly-Pro-Asn-Tyr 7-r--/ From all other data about CB-8, the arginyl of C-26 must be part of T-40. 380 T-4.2 (BBA-18a):

Ala-Arg.

385 T-43 (BBA-19b): Val-Ala-Asn-Tyr-GlnArg. Peptide P-10 described above joins T-

43, T-41, and its tryptic hydrolysis pro382 380 duced Ala-Arg (P-10-T-1) and Val-AlaAsn (P-10-T-2) as added proof oGi=que/nce. 390 T-44 (BBA-4a): Asp-Gly-Pro-Met-Cys395 400 Met-Met-Asp-Asn-Gln-Gly-Gly-Ala-Pro-

405

subsequently were oxidized, there was some evidence for CYA-HSL, that is, methionine was in the sixth position. The following two peptides Th-8

Th-9

Chymotryptzpeptide C-26 establishes that T-41 is C terminal to an arginyl peptide.

395 Met-Met-Asp-Asn-Gln-Gly-GlyI----400 405 Ala-Pro-Asn-(Tyr,Tyr,Pro, Z&Se;) 415 410 (Phe,Ser,Ala,Pro,Glu,His,Gln,Pro, 420 Ser,Ala,Leu,Glu,His, Arg,Thr,His)

were isolated from a thermolytic digest of AE catalase and showed that a methionyl residue occupies the sixth and seventh positions of T-44. It is known that CNBr does not react with N-terminal methionine. Thus, if the initial reaction was with methionine 393, methionine 394 would not cleave. This explains why preparations of CB-11 always contained some methionine and some homoserine. Edman degradation of CB-11 showed both methionine and aspartic acid at the first step. After tryptic hydrolysis of CB-11, the T-44 fragment began mainly at aspartic acid 395 although 0.22 residues of methionine were also present. When this T-44 fragment was hydrolyzed with chymotrypsin, these peptides were isolated: CB-11-T-44-C-l

410

Asn-Tyr-Tyr-Pro-Am-Ser-Phe-Ser-Ala

415 Pro-Glu-His-Gln-Pro-Ser-Ala-Leu-Glu-

420 This 34 residue peptide was isolated in poor yield only from oxidized catalase. From AE-catalase, tryptic cleavage 390 gave Asp-Gly-Pro-Met-AEC (T-44a). The --composition of CB-8 showed that the first four residues belong to CB-8. Therefore, these residues must represent the N-terminal section of T-44. When apocatalase was cleaved with CNBr and the products

409

SEQUENCES

395 Asp-Asn-(Gln,Gly,Gly, x0Ala,Pro,Asn)

CB-11-T-44-C-2

405 (Tyr,Tyr,Pro,Asn)

CB-11-T-44-C-3

405 Tyr-Pro-Asn-Ser-Phe ---7

CB-11-T-44-C-4

410 (Ser,Ala,Pro,Glu,His, 415 Gln,Pro,Ser,Ala,Leu)

CB-11-T-44-C-5

420 (Glu,His,Arg)

His-Arg.

410

SCHROEDER

A tryptic peptide in low yield from a 24h hydrolysis (BBA-2lb) provided the se410 quence of five residues Ser-Phe-Ser-Ala---415 Pro-(Glu,His,Gln,Pro,Ser,Ala,Leu,Glu, 426 His,Arg). Apparently, this peptide arose from nonspecific tryptic cleavage and was actually the C-terminal portion of T-44. Two chymotryptic peptides, C-27

410 415 Ser-Ala-Pro-Glu-His-Gln-Pro7 7 ---:7 Ser-Ala-Leu ---Y=----:7

and C-28

420 Glu-His-Arg-Thr-(His,Phe) -7--

395

Cys,Met,Met,Asp,Asn,Gln,Gly, 405 400 Gly,Ala,Pro,Asn,Tyr,Tyr,Pro, Asn,Ser,Phe) 425 Thr-His-Phe-Ser-Gly-

C-28,described links T-45 to T-44.

Asp- Val-Gln-Arg.

above, 435

T-46 (BBA-8b):

Phe-Asn-Ser-Ala-Asn-

440 Asp-AspAsn-

Val-Thr-Gln-

Val-Arg.

C-29

(BBA-C-9) which was used to complete the sequence of T-45 as BBA-22b in (1) shows that T-45 is followed by a tryptic peptide N terminal in phenylalanine. In CB-11, only T-46 is such a peptide. A peptide from dilute acetic acid hydrolysis of apocatalase provided a larger overlap but was not in good purity. AA-8

430 (Val,Gln,Arg,Phe,Asn,Ser,Ala) 445

T-47 (BBA-23a):

Thr-Phe-Tyr-L.eu-Lys.

P-12 joins T-46 and T-47 together.

450 T-4.8 (BBA-lob):

Val-Leu-Asn-Glu-Glu-

455 Neither chymotryptic, peptic, thermolytic nor acetic acid hydrolyses produced a peptide that showed T-47 and T-48 to be adjacent. However, from 500 mg of maleylated AE-cat, after separation first on four tandem Bio-Gel P-100 columns and purification on Dowex 50-X8 and Dowex 1-X2, one peptide proved to be T47,48 (Table VII). Although no sequencing was done on this peptide, the order must be T-47,48 and not T-48,47. P-12 above and C-30 below prove this point. 456 Gln-Arg.

Lys.

A double tryptic peptide Lys-Arg was also isolated (BBA26a). C-30 and P-13 connect several tryptic peptides in this sector of the molecule. T-50 (BBA-13a):

C-30

Arg.

455 Asn-Glu-Glu-Gln-Arg-(Lys,Arg, ---7’ Leu)

and P-13

430 adequately

Val-Arg-Thr-Phe -7-

457 by

385 390 Tyr-Gln-Arg-Asp-(Gly,Pro,Met, ---:r

T-45 (BBA-22b):

445 P-12

T-49 (BBA-9a):

complete the sequence of T-44. T-43 and T-44 are joined together peptic peptide P-11 P-11

ET AL.

455 Glu-Gln-Arg-Lys-Arg-Leu---_ ---_ 460 (CyS03H,Glu,Asn,Ile,Ala,Gly, 465 470 His,Leu,Lys,Asp,Ala,Gln,Leu)

A tryptic digest of P-13 yielded lowing peptides P-13-T-1

455 Glu-Gln-Arg ---

P-13-T-2

460 Arg-Leu-(CySO,H,Glu,Asn, -----y 465 Ile,Ala,Gly,His,Leu,Lys)

P-13-T-3

470 Asp-Ala-Gln-Leu 7-T

the fol-

which not only link T-48, T-49 and T-50 but also T-51 and T-52 to be described below.

BOVINE

CATALASE

460 (BBA-8a): Leu-Cys-Glu-Asn-Ile465 Ala-Gly-His-Leu-Lys. 470 T-52 (BBAda): Asp-Ala-Gln-Leu-Phe475 Ile-Gln-Lys. The Edman degradation deT-51

termined the sequence of the first three residues of this peptide, and the possibility of either leucine or isoleucine in the fourth residue was shown to be leucine by P-13T-3 above. When the N-terminal aspartyl residue of T-52 was cleaved with dilute acetic acid and the remaining peptide was purified on Dowex l-X2, the complete sequence could be determined. 470

T-52-AA-1

Ala-Gln-Leu-Phe-Ile-Gln--7-y-

and T-59. T-52, or course, is eliminated, and T-59 is clearly the C terminus of the molecule. T-55 then must be adjacent to T-54. 495 T-k6,57,58 (BBA-2Ob): Ile-Gln-Ala-Leu500 Leu-Asp-Lys-Tyr-Asn-Glu-Glu-Lys-Pro505 Lys. Except in long tryptic hydrolysates

in which the -Lys-Tyr- bond is cleaved slowly (and the -Lys-Pro- bond, of course, not at all), these three tryptic peptides are normally found together. Two excellent overlaps of T-55 to T-56 are provided by C-31 and AA-IO. 485

c-31

Ser-Asp-Val-His-Pro-Glu-Tyr7--y--490 495 Gly-(Ser,Arg,Ile,Gln,Ala,Leu, LkG)

AA-10

(Val,His,Pro,Glu,Tyr,Gly,Ser, 495 Arg,Ile,Gln,Ala,Leu,Leu)

475 LYS 476 T-53 (BBA-Sa): Lys. 477 T-54 (BBA-Mb),): Ala- Val-Lys.

470

CB-11-T-52,53 Asp-Ala-(Gln,Leu,Phe, -

475

Ile,Gln,Lys,Lys) 476

Although free asparagine has never been isolated from a tryptic hydrolysate of catalase or its derivatives, peptides C-32 and P-14, however, showed that an asparagine is C terminal to T-58. C-32

for which evidence of linkage through a common lysyl residue is provided by a peptide from dilute acetic acid cleavage of apocatalase. 470

506 T-59: Asn.

500

CB-11-T-53,54 Lys-Ala-(Val,Lys)

AA-9

490

485

Hydrolysis of CB-11 with trypsin for 90 min gave the following two peptides:

-

411

SEQUENCES

475

_Ala-Gln-Leu-Phe-(Ile,Gln,Lys, _ ____ -- -__._ Lys,Ala,Val,Lys)

480 T-55 (BBA-21~): Asn-Phe-Ser-AspVal485 490 His-Pro-Glu-Tyr-Gly-Ser-Arg. Because

of the specificity of dilute acetic acid hydrolysis, data from AA-9 (above) suggest that the peptide after T-54 must be N-terminal in either aspartic acid or asparagine. The only tryptic peptides of CB-11 that meet this requirement are T-52, T-55,

505

_Asn-Glu-Glu-Lys-(Pro,Lys,Asn) -----~ ---:‘- -..; _ 500

P-14 Leu-Asp-(Lys,Tyr,Asn,Glu,Glu, -

-

505

Lys,Pro,Lys,Asn) Residue 506 is designated asparagine rather than aspartic acid because C-32 chromatographed as a neutral peptide on Dowex-1. Further proof that T-59 is the C terminus was obtained from a 90-min tryptic digest of CB-11 from which the following peptide was isolated: 495 CB-11-T-56, (Ile,Gln,Ala,Leu,Leu,Asp, 57,58,59 500 Lys,Tyr,Asn,Glu,Glu, 505

Lys,Pro,Lys,Asn)

412

SCHROEDER

The shorter peptide without the asparagine was also detected. When carboxypeptidase was used to release the C-terminal residue of catalase, several amino acids were released in low yield; asparagine was the most probable C-terminal residue. It was concluded that T-59 is the C terminus of the catalase subunit.

ET AL.

peptides in small yield with C-terminal aspartic acid may be detected especially in shorter hydrolyses. However, normally in 16 h at lOO-llO”C, all aspartic acid will be free. In this period of time, only very minimal cleavage of asparagine occurs. The release of aspartic acid from catalase by 0.25 M acetic acid has been used as an example of this method (16). The 48 h of hydrolysis depicted there did cleave DISCUSSION at some asparaginyl residues as can be The data presented here resolve the se- seen by examining the AA peptides in quences that were incomplete in the pre- Fig. 1. Thermolytic and cyanogen bromide liminary description of the sequence of BLC (2). For example, the initial uncer- cleavages were responsible for providing tainty in the analysis of what is now des- peptides that lead to the final sequence. ignated as peptide T-21 has been answered Considerable information about the latter by the sequencing which places five resi- cleavage had been described (8). The fact dues of aspartic acid or asparagine in this that CB-3, CB-11, and CB-5,6 were one trio peptide instead of the four from the anal- of peptides of 120 + residues, and CB-2, ysis. Consequently, the subunit has 506 CB-6, and CB-8 were another of 50 & resinstead of 505 residues. Although contro- idues complicated their separation and versy has existed for many years over the purification. The short CB-1 and CB-7 were number of subunits in catalase as exam- of minor value and CB-9 (Cys-HSL) and ined by various methods (discussed in CB-10 (HSL) were not isolated. some detail in (2)), both the sequence and the X-ray data (3, 4) unequivocally show Quality of the Peptides and Analyses four subunits. The majority of peptides have been isolated by successive column chromatograProcedures phy first on a cation and then on an anion As already noted, the determination of exchanger, although occasional further this sequence was begun at a time when chromatography on one or the other under peptides were produced almost exclusively different conditions was necessary for puby tryptic, chymotryptic, or peptic hydro- rification. Of course, for the long CNBr lyses. These hydrolysates did, in fact, pro- peptides, separation by size as well as vide a very considerable information on countercurrent distribution and other proa polypeptide which was three to four cedures were necessary. Homogeneity of times the length of most proteins then a peptide was assessed by two criteria: inunder investigation. tegral numbers of residues from the amino Significantly more data were obtained acid analysis and a unique N-terminal resfrom the cleaving out of aspartic acid with idue by Edman degradation; examination dilute acetic acid. Although use of a sim- by paper chromatography or electrophoilar cleavage has frequently been reported resis was not done. The peptides in Tables in the literature of sequencing, most work- II through VIII have varied in purity from ers use dilute hydrochloric acid (0.03 N) excellent (P-9, P-10, etc.) to poor (C-15, Cwith the attendant cleavage not only of 19, etc.). Peptides with these poor analyses aspartyl residues but also at bonds N ter- are used because they best illustrate the minal to seryl and threonyl residues. We sequence in Fig. 1. Misinterpretation of have employed the original conditions of data is unlikely because of much addiPartridge and Davis (15) with 0.25 M acetic tional information that is not presented. acid and have found it to be very specific The Edman degradation often identified for aspartic acid. The first cleavage clearly the contaminant; thus, C-19 contained is on the carboxyl side of aspartic acid and Asp-Leu-Phe (Residues 263-265).

BOVINE

CATALASE

SEQUENCES

413

Low values for tyrosine in early analyses were obtained prior to the addition of phenol to the acid. Although values for tryptophan in catalase itself were satisfactory with barium’hydroxide hydrolysis, CNBr peptides which have passed through many stages during isolation gave erratic data.

of 5.3 acetyl groups per 250,000,” mass spectrometry does not show an N-terminal acetyl group. In retrospect, it is possible that the acetyl groups in these preparations derived from incompletely removed acetate rather than from covalently bound acetyl groups. The X-ray data do not aid in identifying the blocking group.

Quality of the Overlaps and Validity of the Sequence

Sulfhydryl and Disulfide Bonds

As previously noted, the data that have been presented are the minimum necessary to establish the sequence. For the most part, the overlapping sequences from various cleavages and additional available data leave only an unlikely possibility for error in those sections of the molecule. Nevertheless, there are other sections where the joining of fragments has been done by elimination or indirection. However, increased confidence in these conclusions comes from the X-ray data because Murthy et al. (4) state that “The structure was in complete agreement with the amino acid sequence with most side chains being clearly visible.” Dr. Michael Rossmann has kindly examined the X-ray data at sequences where elimination or indirection lead to the final sequence. These were residues 45-50, 104-106, 151-154, 167-170, 1’77-179, 210-214, 276-280, 361-364, 391394, and 455-457. The sequence and X-ray data are in agreement except in the last two instances. For residues 391-394, the fit is not good; this is a site of heavy metal attachment and the noise in this area may be responsible. Residues 455-457 cannot be checked because of technical aspects of model building. The inability of the X-ray study to detect the N-terminal four and C-terminal six residues which are definitely placed in the sequencing work probably is due to disorder in the crystal form. Consequently, it may be concluded that the proposed sequence is close to reality. The N-Terminal Blocking Group Despite attempts by mass spectrometry, the N-terminal blocking group has not been identified. Although it was previously reported (1) that different preparations of catalase had “4.3 to 6.4 with an average

The cysteic acid content of oxidized catalase and the AEC content of reduced, aminoethylated catalase are indicative of four half-cystine residues per subunit (Table I). Are inter- or intrasubunit disulfide bonds present? The evidence against intersubunit disulfide bonds is clear. The sequence and X-ray data show four subunits. These, however, cannot be joined by disulfide bonds because ultracentrifugal data under denaturing, nonreducing conditions show subunits of the appropriate molecular weight (17-19). Although oxidation or aminoethylation under reducing conditions did show the presence of four half-cystine residues per subunit, reaction with various sulfhydryl reagents such as ethylenimine, vinylpyridine, etc., under denaturing, nonreducing conditions revealed only 2-3 sulfhydryl groups per subunit. These chemical experiments are now of academic interest because the X-ray data show that the distance between any two sulfhydryl groups is too great for disulfide bond formation (4). In the accumulation of X-ray data, cysteinyl residues 376, 392, and 459 formed heavy metal derivatives, but Cys 231 did not and is considered to be totally buried. On the other hand, the possibility cannot be ignored that Cys 231 may be in disulfide linkage to some small molecule as in streptococcal proteinase (20). The C Terminus According to the evidence presented, asparagine at residue 506 is the C terminus of BLC. Murthy et al. (4) mention, on the basis of information that was provided from this laboratory, that a small fraction of the subunits may have 10 to 15 additional residues at the C terminus. These were observed in a thermolytic digest of

414

SCHROEDER

a single sample of commercial BLC during the final stages of the investigation. The amino acid compositions are given in Table V as peptides Th-10 and Th-11. They are assumed to arise from an extension at the C terminus because no place can be found for them in residues 1 to 506. When another production lot was examined, only a trace of Th-11 and none of Th-10 was detected. Furthermore, when BLC was isolated from fresh liver, neither Th-10 nor Th-11 could be isolated from this product. The sequence of Th-11 was Val-HisThr-Tyr. BEC was then examined. Not only were peptides with the same amino acid composition as Th-10 and Th-11 isolated, but with the composition also another (Val,Tyr,Thr,His,Ala) which presumably is related to Th-11. The quantitative relationships of these peptides to the other thermolytic peptides of BLC and BEC differed. The yield of these peptides from BEC approximated that of the average of all thermolytic peptides but was only about a third as much as the average thermolytic peptide from BLC. It was concluded that this extension at the C terminus is normal in BEC, but occurred in only one of three or four chains of this particular lot of BLC. Several explanations for these observations may be made. Consider the quantitative aspects of these peptides in BLC. Suppose an abnormal state occurred in a particular bovine liver so that all subunits of BLC had the C-terminal extension. However, assume that in the normal situation there is no extension in BLC. If, in the isolation of the BLC, the abnormal liver and two or three “normal” livers were used, the BLC with extensions would be diluted. But why should BLC and BEC differ in this way? The milieu in liver and erythrocytes differs greatly. It has long been known (21) that liver catalases do not have a unique prosthetic group, but one which is a varying mixture of hematin and an iron-biliverdin complex. Erythrocyte catalases, on the other hand, have a hematin for each subunit. The activity of a liver catalase is in direct relationship to the number of hematins in the four-subunit molecule (the topic is reviewed in (22)). The iron-biliverdin complex is

ET AL.

thought to derive from the oxidative degradation of hematin. Perhaps what is normally observed in the liver is a molecule in the first stages of catabolism; not only is the hematin partly degraded but some of the protein moiety also has been removed. Another possibility that may be considered is the processing of the protein moiety after biosynthesis. It may be that normal processing removes this extension in the liver but not in the erythrocyte. Histidine

7.4at the Active Site

Margoliash and collaborators (23, 24) studied the inactivation of catalase by aminotriazole. By tracer with radioactive aminotriazole, they were able to isolate and characterize a tryptic peptide which when correlated with the sequence data identified His 74 as the reactive residue. The X-ray data (3, 4) indeed place His 74 on the distal side of the heme group in a position to participate in the catalytic activity of catalase. Bovine Erythrocyte

Catalase

The examination of BEC was made in order to determine whether the catalase in two organs of the same species differed in sequence. The method of isolation of BEC is described in the supplementary material. Table I has an amino acid analysis of BEC. The peptides in a tryptic and a thermolytic digest of BEC were isolated. These peptides were purified and analyzed and the Edman degradation was applied in the same manner that has been described for the peptides from BLC. It does not seem of value to provide tables with all the data from amino acid analyses of peptides, etc. Such information may be obtained from the authors. The result of this examination of BEC has been to substantiate the data from BLC. Except at the C terminus as described above, the sequence of BEC is identical with that of BLC. The amino acid composition of these peptides from BEC account for 493 of the 506 residues of BLC. To the extent that the Edman degradation has succeeded with each peptide, 298 residues may be placed identically by analogy

BOVINE

CATALASE

to BLC. Those residues that have not been accounted for in BEC are 14-15, 65, 136143, 210, and 506. As noted, the peptides equivalent to Th10 and Th-11 were isolated from BEC also and in addition one with the amino acid composition (Val,Tyr,Thr,His,Ala). The peptide with the composition of Th-10 had the sequence Val-Gln-His-Gly-Ser-His. Although theyeGn= r (Val,s, Thr,His,Ala) could not be determined, it presumably is related to Th-11 and may have the sequence Ala-Val-His-Thr-Tyr. This seems probable because human erythrocyte catalase definitely has alanine as a 507th residue and may have as many as 520 residues (25). These presumed 11 residues at the C terminus have three histidy1 residues. Residues l-506 have 21 histidy1 residues per subunit. Consequently, the amino acid composition of BEC should reflect this increase in comparison with that of BLC. However, it is obvious from Table I that this is not so. Although the sequence data for BEC are incomplete, it seems reasonable to conclude that the catalases from bovine liver and bovine erythrocytes are identical in residues 1 to 506. It would appear that at some point in its history, BLC also has the larger number of residues but the lesser number in BLC as commonly isolated may be due to different processing of the molecule or to its partial degradation. ACKNOWLEDGMENTS We acknowledge with thanks the assistance which the following individuals have given to one or another aspect of the investigations: Dr. Heinz Boettger, Dr. Costello Brown, Dr. Joyce Chang, Mrs. Jean Cormick, Mr. Johnson Cua, Mr. William Fenninger, Mr. Philip Lieberman, Mrs. Barbara M. Olson, and Dr. Manfred Renz. Dr. Michael Rossman kindly provided data about the X-ray crystallographic results prior to publication.

415

SEQUENCES M. G. (1981) Proc. Nat.

Acad. Ski

USA 78,

4767-4771.

4. MIJRTHY,M. R. N., REID, T. J. III, SICIGNANO,A., TANAKA, N., AND ROSSMANN,M. G. (1981) J. Mel Biol. 152, 465-499.

5. SCHROEDER,W. A., SHELTON,J. R., ANDROBBERSON,B. (1967) B&him. Biophys. Acta 147,590592.

6. HIRS, C. H. W. (1956) J. Biol. Ckem 219,611-621. 7. BRATTIN, W. J., JR., AND SMITH, E. L. (1971) J. Biol. Chem. 246, 2400-2418. 8.

SCHROEDER, w. A., SHELTON,J. B., ANDSHELTON, J. R. (1969) Arch. Biochem. Biqphys. 130,551555.

9. SCHROEDER,W. A. (1972) in Methods

in Enzy-

mology (Hirs, C. H. W., and Timasheff, eds.), Vol. 25B, pp. 203-213, Academic New York.

S. N., Press,

10. SCHROEDER,W. A. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N. eds.), Vol. 25B, pp. 214-221, Academic Press, New York.

11. NOLTMAN,E. A., MAHOWALD,T. A., AND KUBY, S. A. (1962) J. Biol. Chem. 237, 1146-1154. 12. SCHROEDER,W. A. (1972) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N. eds.), Vol. 25B, pp. 298-313, Academic Press,

New York. 13. SCHROEDER, W. A., SAHA, A., FENNINGER,W. D., AND CUA, J. T. (1962) B&him. Biophys. Acta 58, 611-613. 14. LIU, T. Y., AND CHANG,Y. H. (1971) J. Biol. Chem. 246, 2342-2848.

15. PARTRIDGE,S. M., ANDDAVIS, H. F. (1950) Nature (LmAm)

165, 62-63.

16. SCHROEDER,W. A. (1968) The Primary

Structure & Row, New York. 17. TANFORD,C., AND LOVRIEN, R. (1962) J. Amer. Chem. Soa 84, 1892-1896. of Proteins,

p. 110, Harper

18. SUND, H., WEBER, K., AND M~LBERT, E. (1967) l&r. J Biochem 1,400-410. 19. SAMEJIMA, T., MCCABE, W. J., AND YANG, J. T. (1968) Arch B&hem. Biquhys. 127, 354-360. 20. FERDINAND, W., STEIN, W. H., AND MOORE, S. (1965) J. Biol. Chem 240, 1150-1155. 21. LEMBERG,R., AND LEGGE, J. W. (1943) B&hem J. 37, 117-127. 22. CHANG,J. Y., ANDSCHROEDER, W. A. (1972) Arch. Biochem

Biophys.

148, 505-508.

23. AGRAWAL, B. B. L., AND MARGOLIASH,E. (1969) Fed Proc. 28, 405.

REFERENCES

1. SCHROEDER,W. A., SHELTON, J. R., SHELTON, J. B., AND OLSON,B. M. (1964) &o&m. Bio-

24. AGRAWAL,B. B. L., MARGOLIASH,E., LEVENBEKG, M. I., EGAN, R. S., AND STUDIER,M. H. (1970) Fed. Proc. 29. 732.

25. SCHROEDER,W. A., SHELTON, J. R., SHELTON, .I. B., APELL, G., EVANS, L., BONAVENTURA,J., 2. SCHROEDER,W. A., SHELTON, J. R., SHELTON, ANDFANG,R. S. (1982) Arch B&hem. Biophy~. J. B., ROBBERSON,B., AND APELL, G. (1969) phys. Acta 89, 47-65.

Arch. B&hem

Biophys.

131, 653-655.

214.422-424.

3. REID, T. J. III, MURTHY, M. R. N., SICIGNANO,A., 26. BONAVENTURA,J., SCHROEDER,W. A., ANDFANG, TANAKA, N., MUSICK,W. D. L., AND ROSSMAN, S. (1972) Arch Biochem. Biophys. 150,606-617.

416

SCHROEDER SUPPLEMENTAL THE

COMPLETE

AMINCI

CATALAsE

AND

THE

ACID

MATERIAL

A.

Schroeder,

Barbara

PARTIAL

MPERIMENTAL

product

source

of C. F. Boehrmger

Handling

of this catalase

from

this

were

c”“c”rdant.

material

was the commercially

und Soehne,

prior

as we,,

Marmheim,

prepared

Shelton,

Joan 8.

and Gerald

Ape11

preparat,ons

vided

the chymotrypttc

all

An 0.8-g

(1).

from

fresh

Results

p”

liver

6 Wh

hens

Chenucal

tured

catalase,

most

tzon to the chemxal wth

procedures

but Ular

preparab””

products

d,d not ymld

The

because

of heme

descnptmn

of H,rs

(7).

After

followed

acetic

acid,

dark-colored

soluhon adjusted

at pH 3.9.

formed

8.5 and 9.5 through

was transferred

the sample

remo~l

pH 9.5.

After

E HCI

9 g. N&H

I” small

tubing

and three

to

m 7.5 ml

1 g of malex portion.9

suspension.

to dialysis

dropwise

to 40 ml with

After

and dialyzed

changes

(2.2

Substrate m water Durmg

lower

After

react,on

removed,

suspended

(normally

p” mm-7*7

added

and the p”

adjusted.

3.5)

Usually,

equal

(2.2

15 min,

However,

and many

were

Sometimes,

w-as cenblfuged

w-as lyophihzed

for which

the sample

after

at 40 C for taken

material imtially

solvent

for

a

24 h at

as short

was 0.2

50-X2

only

lo 2.05

B wade,

WIS added.

After

directly

to R Dowel

g) was Suspended

wth

IoL

18 b at !‘oom 50-X2

column

I” 40 ml of water

(CalBmchem

was apphed

Lot No.

was added

directly

of peptades.

of small

000959,

at 0 and 1.5 h.

to 1.9,

prec~p,tate

to a Dowex

papain

M KCN,

34 h at 36°C

water

were

50-X2

was

column

sample

I” a stream

of pH to was done

of .ur and

200 ill of water

aeetate,

(15 mg/ml).

The

p”

5.2,

sample

to

20 ,A of

was incubated

to chromatography.

(IAP)3.

20 ~1 of 0.025

To the peptide

M M&h,

solution.

was terminated

were

M sodium

prior

ammopeptidase

adjustment

and incubation

the mgredtents

and lyophilized

and

done in a test

for chromatography.

20 p, “I 0.2

and 20 pl of LAP

After

was then dr,ed

solvent

chymotryphc,

normally

was added,

and 10 ~1 of papam

the reaction

were

as a tufler.

hydrolysis,

added

Tryptic,

peptides

NH$CO,

The

the sample,

0.1

20 ~1 of 0.5

After

by removing

m 200

111 of

M tris

buffer,

incubation

at 40” for

the solvent

in a stream

16 h, of air.

Cm

for

All columns

tryptic

dzgests

water Both were

!3epamu”n

catalase flask, sample

Optimal (6).

temperabwe

Amd.

This

and 500 ml of 0.25

acid pH 3.1.

chromtopraphy

N acetx

and again

for Dowex

60-X3

added,

were

have dissolved

and the solution After

the sample

ten-fold

dilution

v,as dtsaolved

and

and cO”nterCurrent

of the CNBr

for the selective

cat as follows:

was reflued

was oxidized

clewa@

pepttdes

after

the

(Bee bel”w).

procedure

was carried

the solutmn

pH 3.1 buffer

were

chromatography

by gel fil~auon

CNBr3

in the dark.

and ly~~hilizatioq ton excha”Se

for

MM mg of AE-cat

used for purification

residues,

(see below)

conditions

Typically,

300 mg of CNBr

16 h at room

Acetic

to

in detail

relyophllized.

aspartyl

Any precipiby procedures

Bromide.

in 12 ml of 10% TFA3, stirred

iniw

to pH 2, the sollltio”

M pyridine-acetic

(0.6

mf of enzyme

16 h.

for

or when pyridine-

then redissolved.

of

I” 30 ml 01

ad,ustn,ent

3X tryst,

to 1% 01 substrate

up in the appropriate

~4th dis”“ed

as

of the precipihte

subsequently

on Dovex

0.5

been presented

was

hydrolyses

formed.

adjustment

equal

n11 of 0.59,

dirrtribution

precipitate

E “Cl

dqestmns

Cyanogen

made.

off and examined

and tie

were

most

rolumn

at

at pH 6.0 wti

the short

p”

Thermolysm

formed

weight

to proceed

when pH w-as reduced

was added,

Gccaaiodly,

chromatographed

For

was tissolved

was dxssolved

(CalBmchem,

to 8.0.

d,gestlons

8 if necessary,

(Worthington

of substrate

of the hydrolyses,

acid developer

he described.

were

winch

Trypsm

hydrolysates

done at 4o’C.

of trypsl”

At the conclusion had ditlsolved.

some

d solution

the precipitate

was allowed

temperature

x 100 cm).

pH 8.5, be

of

if the substrate

However,

to 0.5%

stirring. (It should

unsatisfactory

satisfactor,ly.,

the hydrolysis

one or two additions

were

was done,

or TRL-6227)

overnight

occurred.

were

d,gested

room, temperature.

Ute

with

or Suspended

of O-cat

for 2 h at 37 , the pH was lowered

Leucine

at 0, 3, and 10 h, and the pH was mamtained

pH-stat.

acetic

mg/ml

precipitation

dgestmns

about

ad]ustment

wth

the

column

The pH was taken

on n 3.5 x 100.cm

was npphed

ard the sample

tube m 0.5

was digested

0.1 to 2 g was dxsolved

of IO-15

of pH to 6.0,

that enzymatic

p”

or a derivahve

w,th

at room

Followw

AE-cat

adlusted

65’; E, 31’; CaAcl)

was as follows.

from

at a concentration

merely

durmg lots

in amou,,f

adjusting

remarked were

apocatalase

procedure

at addl-

x 100 cm).

For

Whether

the general

rng of pepsin

!,,e sample

o”er

solu-

d,luted

and the material

st,rrm~.

108814) I” 1.3 ml of 0.01

was clear.

Trypsln.

7.5

Enzymatic

against

to 6.5,

was dqsted

sample

much

No,

dissolve

trypan,

A 0.7-q

N WI,

thermolytx

of dialysis

temperature

for 3 h wth

at 0, 3 and 10 h.

was lyophllzed,

0.5

anhy-

15 minutes

at rwm

on a 3.5 x 100~cm

for chromatofraphy

with

and thr p”

into a mull

pro-

IV.

DOWeX 50-n.

the pH was maintained

was added stirred

24 hours

was added

While

of

made

was Qluted

of 2 N NaOH,

and Bell)

to the constantly

which

to a. 2 with

additzon

Coleman

01 1 hour

were

3 ml of water

a precipitate

(Mafheson,

the deacriptlon

500 mg of AE-cat’

As he pH was being

tion,

without

of AE-eat of enzyme

Thermolysm. procedure

of H,G,

NH,,

the solution

Pepsin.

I” (1).

or AE-cat

Table

lot 6003)

chromatorraphed

sample

3.1 buffer

of the

ol the ammoethylatlon

(6) at -10 to -20”

water.

the suspension

the examination

was done as desenkd

a clear,

0.2%

made,

was digested

The pH was lowered

additions

temperature,

,nethod

This

10 ml of glacial

dride

also

to 2.1, m p”

0.01

produce

a period

were

for 34 h wth

used.

and Smith

between

and benz,,

In addImOd,ficatlons

I” (5).

Maleykation.

with

below,

mformltlon.

A detaled

15 gven

OxAat,on.

Brauio

dwlfide,

Rem”~,

Am,noe,hyht,on.

MS

done w,th apocatalase.

1s not described useful

of heat dena-

listed

(Wortiington

done in much

of O-cat

that are

of O-cat

bulfer,and

A 1.8-g

d,gestion

to be descnkd

carbon

Apocatalase.

Of apocatrdase

were

try,,t,c

peptides

portion

were

Digests

50-x2.

o, BLC

used

mod,ficatmns

SUCCI~,C anhydrlde,

of chloride

Mod,f,catlons

hydrolyses

hydrolyses.

1% a-chymotrypsin

at 0 and 2 h.

Dowex

a Iew exper,ments

Chymotryptic

way as tryptx

appropriate

AWwgh

Shelton,

the same

Germany.

to use has been described

as from

LIVER

Chymotrypsin.

of BLC

of BLC

FJcJvINE

OF BOVINE

CATNASE

PRGcEDLmES

Source The pr,mary

OF

SEQUENCE

J. Roger

Robberson,

To

SEQUENCE

ERYTBROCYTE Walter

ET AL.

cleavage

After

lyophilized. chromatography col”m”.

in a I-bter

lyophllization.

Material

of

500 mg of apo-

ac,d had been placed

for 48 h.

on a 1.4 x 100-m,

After

msoluble

wds removed

the m the prior

to

BOVINE Formic

Ac,d.

preferentially

In order to cleave the aspartyl-probne

m peptide CB-3,

70% formic

acid and maintamed

of H,O and lyophlhzatmn

250 mg were dissolved at 50’ for 24 h.

CATALASE

SEQUENCES

bond Amino acld analyses

m 50 ml of

Acid Analyzer

D~lutmn wtb 500 ml

long column and flow cells wtb mcreased

to dryness followed.

Hydrolyses

were normally

doubly glass-dmtilled With the exception countercurrent cation-

of snrne separations

distiibubon,

or mm-exchange

,,horesw

although

by ge, Illtraha”

peptide separatmns chromatography.

catmns were done by paper or thin-layer

were achvwed

No isolahons chromatography

color tests for tryptophan

or

protein

by

hon.

determined

ion-exchange

Chromatography.

An example of the separatmn

pepbdes of catabase is given in (1).

rations

on the cation exchanger exchanger

AG l-X2 in (lo).

separated

0.6 x 60 cm).

matographed

Detailed

descriphon

01

destruc-

2 drops of 5% phenol were added

Of tyrosine.

in eatalase and in cyanogen bromide

with barium

hydrtide

fragments

with some modincation

heating at UO” for 64 h & =.

of sepa-

et&.

Dowex 50-X2 has been given m (9) and

other digests were chromatographed of Dowex 50-X2 rest. Tbe simpler were further

of the whole

of hydrolytic

%-as

ol the method

of Noltmann --et al. (11). Because Vycor test tibes are difficult to seal, they were inserted mtO a larger Pyrex tube titch was sealed prior to

tryptic

(usually

a calculatio”

In some of tbe later hydrolyses, the recovery

path length. Some hydrolyses

times to per”,,,

Tryptopha”

were done after paper

Beckman Ammo

made for 24 h at 110” m 2 ml of

6N HCI $ s.

used l.,“ger

to nnprove

or purifior electro-

cbromatagraphy.

on the mm

were made vntb a modtiled

that had a 0.9 x 6-cm short column and a 0.9 x W-cm

Routinely,

alter evaporation

or

on smaller

sbl, existed,

Ref. 9 provides

a detailed

gradients,

cross-linking,

etc.

discussion

in a rotmy

evaporator,

ment of pH, d necessary. Tryptophan development of the long column rather

AG 1-X2 colllm”S

on a 0.9 x 60-cm colum

they were again chro-

followed Noltmann the barium,

and

the sample was not filtered with adjust-

was determined wit,, a mcdtfied than 0” the short column. Thus,

of Beckman AA-15 resin titer

development

at

pH’s 3.25 and 4.25, change was made to a third buffer (short column,

on Dowex 50-X2 or X9 (0.6 x 60 cm) with a modified

gradient.

work-up

but faken up m pH 2.2 buffer for the amino acid analyzer

first on appropriately-sized columns mixtures from these chromatograms

by chromatography If mixture8

large tryptic

Further

(11) except that dry ice was wed to precipitate

pH 5.28 buffer and “-propanal

of the effects of modified

emerged.

Tryptophan

in 9:l raho) mmwdtately

eluted approximately

alter

leueine

60 mm after phenylakmine

at a flow rate of 66 ml/h. Prolme, valine, Iwcine, and phenylalanine all survived basx hydrolysis and could be used to correlate basic and ac,d,c hydrolyses

of the same sample.

Tryptophan was detected 1” small peptides after paper eleetro,,hores,s or paper chromatography by spraymg the ninhydrin-reacted spots wth cmnamaldehyde reagent (0.4 g cinnamaldehyde 951 ethanol and 10 ml of SE HCL). Countercurrent further ho”.

separatmn The detals

D,str~butmn.

Th,s metiod was zpphcd lo the

done m 0.5 N acetic aad.2.buta”ol:,O’; of 1O:Q:l.

s All Edman degraddtmns

of CNBr peptIdes 3, 4, 5, 6, and 1, alter ire, f,,traha”e bee” described

For red,stnbuhon

phase, 3% tnchlor~acebc

(8).

The ,mt,a,

drchloroacetx

dzstnbubon

the PTH-ammo

ws

,n the aqwous

,dent,f,ed

(12).

Asparaglne

paper strips and

Details

and glutamme

of the procedure were directly

I” this system.

ac,d and 0.5 N forrmc

In certam

mstances,

a procedure

was apphed only once or

~,V,CE to solve a spec,f,c problem ,n sequencmg. These procedures are described I” the text where the appllcatm” was made.

acid.

THE ISOLATION

directly.

Other Procedures

acid re,,laced IO’; diFor pephdes that ch,oroacet,c ac,d and 0.5 N xetlc ac,d respectwely. moved rapldly ultb the or@mc phase, 58 monochloroacehc ac,d replaced 108 dwhloroacebc

were done on filter

acid was ,denbf,ed

have been described

ac,d m tie rabo

of pe,,hdes that were largely

in 90 ml of

AND PROPERTIES

OF BOVINE ERY~-HROC,‘TE

CATAWSE Walter

A. Schroeder,

Rxhard

The ~.olat,ve procedure for human erytbroeyte catalase (HECj3 Much has bee” described (26) requires some modification for “w LSOlatm”

of BEC.

The fo,,owng

descriptm”

notes only those aspects of

the mxlahon which differ from those for HEC. Where no comment is made, reagents, relative quantities of soluhons and reagents, procedures,

etc.,

are Identical. sovrce Of Blood

Bowne blood was obtained nnmediately slaughtered.

As anticoagulant,

S. Fang, and Joseph Bonaventura

w With hum.n er~throcytes, hemolysts IS followed by deionization with mixed bed rest. With bavlne erythrocytes, deionization did not proceed well unless the bemolysate

in tbe refrigerator after tbe animal

30 mg of heparm in 100 ml of 0.9%

small clots were removed and the blood was tubbled witi

before the supernatant

was

NaCl was mued with each 4 Uters of blood. The containers were s”rrounded by ,ce for transport to tie laboratory where any foam and Co to convert

to carbanmonoxyhemoglob,“. WJ Wdh human erytbhrocytes, packed cells were available and mrxmg with sucrose sohtmn caused the cells to clump together and settle rapidly. With bovine blood, the same effect MS observed but a Ima, BEC o, adequate purity could not be obtained ualess the bvlne cells were packed at 6000 g for five minutes, then mtied with 7 volumes of sucrose so,“tion, and allowed to settle.

was treated

with CHCI, equal to

0.1 the ongina packed cell volume. After sttrring for 10 tin vntb a large propeller stirrer, the CHCI, was allowed to settle for 20 min solution

was siphoned off.

v Deionizatmn, and batchwise adsorption follow exactly the procedure for HEC as does the mittal washing of the CO,““I” with 1.5 mM phosphate b,,ffer prmr

to desorption.

The desorption

used 30 “04 I”-

stead of 20 mM phosphate bufler. I” “,e BEC ~solatmns, the column prior to and after dewrption tended to have more fued brovmisb-red material (presumably ferrihemoglobin) possibly because of the treatment with CHC&. m” somewhat dxkTent percentages Of saturation with (NH,),so, are necessary wth BEC. At pH 7.0, the concentrahon IS InsL taken to 30% for 6 h; the white material IS centrtfuged off and discarded. The

418 main

SCHROEDER

crop

surring

of crystals

Is produced

an* f”rther At p”

In contrast solved

to Z&30%

lor

BEC

crystallizes

over

very

of the pro(1res8 dark

clpitate

green.

green

bxlicati”e

final

A2&A405

ratio

Consequently,

Approximately

the (NH,),sO, should

liver

cells yields

Such a Lye,

The progress raha.

Thw

stages,

cat

commented

may be tan to brown

for the next

till

of the presence

should

Of some

these

or

operation

Am,“”

acid

Aetd Compos,ti”n

of Cyan”@”

and may by

hemoglobin.

HOW-

At”,“0 And

TypIcal Arlalyslaa

Amino acid Trpb LYE AEC 2.9(3) X0(3) ;:y’ 3.w 2.1(Z) 2.0(Z) 2.m 0.1 2.0(Z)

0.1(l) 4.0(4) 0.5(l) 3.ow 2.2(Z) ‘i-i I if’ 2:7w 4.96) 2 I ii; 6: O(7) 4.0(41 2.9(3) 5. B(6) 5.1(6) ;: $1 0.3 72

Numbers

I” parentheses

Peptides.

0.5(l) 1.9(21

a liter

of

a kg “1 bovine

of BEC

IS given

in Table

I and

extinction cm-’

coefficients

of BEC at 260 and 405 nm

and 4.2 x 105K1

is 0.79.

cm-‘,

The wavelengths

A molecular

we,gbt

respectively.

of maxim”m “l 2.31

The

absorption

are

x IO5 is assumed

for

data. vel”city

The heme

content

In spectral

da%

constant

was 3.3 x 10’

hter

by the pyridtne-bemocbromo~en

M-’

sec.‘.

procedure

was 3.6.

comparable

BLC From sequence

to HEC

specific

aetwtty,

and heme

content,

BEC

is

(26).

& Llteragre (13) 27 1 :i 69 24 34 46 39 36 36 35 to 19 36 22 31

we,@f

LO(l)

1.0(l) 0.9(1, ,.1(l) X9(4) 1.1(i)

1.0(1)

2 :I$ 2.1(Z) 5.W 0.W) 0.3(i) 0.8 0.7 55

from

a

LOW ::3 2.0 I 2) 1.7(21 5. S(5) 6.5(71 2.4(Z) 3.2(3) 5.0(5)

may be is&ted

on in the Discussion.

The standard

in the s”OI”tmn

Br”mlde

The

0.16.

of BLC.

composihon

and the following

wll

be followed

and Ule

proteins.

to 280 mg of HEC (28) whereas

a gram

at 276 and 405 nm.

pre-

=24-h hydrolyw, bC”rrected to 232.090 molecular and dwxded by 4. CA”erage Of 7 analyses. dmm other ana,yses: see text.

U:

about

ratio

25.8 a.dd 20.5 30.5 70.6 21.6 23.1 48.0 37.1 34.1 36.4 34.1 9.9 17.0 34.3 19.7 31.1 5. zd

Table

in contrast

3.3 x lO%i’

A2so/A~5

~111 be 3.

fractions

be meamngless

were

mdi-

the centrifuged

ol the purll,eatto” ratio

product

the tan to brown

that is used

the A&&s

because

The final

at other

Often

IS a ww!

of hemoglobin

of non-heme

approximate

60 mg of BEC

The amin”

be

1s centr,f”Ced

precipitate

be free

B

material

s”l”tion

any matenat

sbculd

18 dis-

after

tie

shwld

Of the presence

The molecular

green.

in the solvent

desorption

saturahon

tan to brownish

concentration,

However,

be discarded.

from

slowly.

of the purificabon.

and dark

be insoluble

at 6-95,

precipitate

becomes

packed

of any centrifuged

may be layered.

Ughter

occurs

the (NH&O.

ratio

is added.

The appearance

tiorm

sane

of 6 to 12 h bef”re

(NH,@J,

ever,

off.

to an approprrate

periods

12 h cd

When the pH 1.0 precipitate

and was centrlfu~ed

ba6 been taken

eator

cd EC

HEC.

with

a, 40%.

t” the pH 4.6 precipitation,

‘*pa tnmluble

and m”re

at 38% saturation

Crystallize8

4. S, the cryehllizatlon

prtor

rtirred

mater1a1

ET AL.

11

are tie reddues

0.3(l) ,.9(Z) 5.7(6) 5.0 5) 0.9 I 1) &O(3) 5.0(5) X0(3) 3.1(3) 2.1(Z) 0.1 1.00) 4.0(4) 3.5(4) 1.0(l) k;(l) 42

of each =“I”,”

(1)

(1)

9.5(9) 0.9(l) 4. a(5) 5.9(6) 1::; 5’ I 6.1(7) 16.3(16) 5.0(6) 5.6(5) 8.4(8) 6.4(B) 0.3 3.1(3) 7.8(9) 4.5(5) 5.9(6) (11 0.1

2

acid from

1 112

Le sequence

(1) .x0(4) 1.6(l) 3.1(3) i: : I :y’ 1.4(Z) 6.503) 5.2(51 6. S(2) 3. r(4) 5.6(6) 0.5 5.2(5) 1.3 1.7(l) ii. D.(Z) 0.5 60

data.

&O(6) 0.w 5,8(?)

5.4(B) 0.6(l) 3.8(4)

1.4(l) 0.6(l) 2.0(Z)

0.3(l) 3.0(3) 2.1(2) 3.1(3)

2z&

1;g

iig

12.4(12) 10.9(11) 10.4(10) 10.6(10) 10.8(11) 0.2 &O(6) 13.3041 7.4(E) 11.0(12) 0.6(3) 1.6

lo:s(lo) 9.4(9) 6.3(8) 0.6(10) 9.4(9,

4:0(4) 6. S(9) 4.0(4) 3.0(S) i:;(2)

3.3(3) X4(4)

4.7(5) 10.4 11) 6.6 8) 9.0 9, 0.62)I 1.0

2.0(Z) 3.3(4) X9(4) 1.5(l) 0.6(Z) +

3.0(S) 3.94) 1.1 I 1) 5.5(6)

3J

158

121

53

91.g 3: 4(Z)

50

BOVINE

PeptIde Residue

P-l I-26

rm8.

P-3 51-53

P-2 41-50

P-4 51-59

P-5 110-112

CATALASE

P-6 154-159

P-l 249-281

P-8 308-315

P-9 332-354

0.80)

1.0(l)

1.0(l)

G(l) 1.0(l)

0.2

1.0(l)

1.10)

2.2(Z)

0.1

0.1 LO(l) 5.015) 0.8(l)

l.O(ll

0.1

1.1(l) 0.8(l)

0.9(11 4.0(4)

0.3 1.1(l)

2.m 0.4 3.9(4) 1.3(11

0:1 1,1(l)

0.2 0.2

:.:I:; 1: Z(1) 2.6(3)

:: iIS

2.0(2) 1.2(l)

5.1(5) mt2;

0.1 1.0(l)

LO(l) 0.9(l) 0.9(l) 2.0(2)

LO(l) 0.8(l) 2.2(2,

LO(l) 2.0(Z)

2.1(3)

2.1(21 0.9(l)

C-l 18-26

c-2 51-63

c-3 64-14

c-4 15-81

c-5 66.93

C-6 94-99

LO(l)

0.1

0.2

1.00)

0.1

2.0(Z)

0.9(l)

l.O(ll 2.9(3)

1.2(l) 0.941) 0.9(l)

0.m

1.0(l)

%, 0.2 0.4 1.2(l) 0.2

i:+;

l.?(2) 0.1 1.00)

1.1(l)

1.1(l) 1.0(l) 0.2 2.0(2) 0.9(l)

“0%

P-1$ 453-411

P-14 498-W

a.1 2) 1.1 1)

&l(S)

E I :; 5:3w X5(3) Z(l,

2:; I

2.11(a)

2.3(Z) 3.1(3)

:: i{S 2.0(2, 2.0(Z) 1.0(l) l.O(ll 0.8(l)

;:A;/

0.3 2.3(3) 1.2(l)

1.00)

0. T(l) 0.1 0.2

0.2 0.a 3.9(4)

0.2 0.1 LO(l)

1.21) 2.0 I 3) 0.2 1.0(l) 2.9(3)

0. I

2.8(S)

;.; ;j .I 0.4

LOW 0.8(l)

LO(l)

(1)

0.80)

PeptIde Residue

P-la 443-445

1.1(l) El:; 2.0(Z)

1.0(l)

ncx.

P-11 385-408

0.2

LO(l)

yw;

1.1(l)

Peptide Residue

P-10 370-384

f: $321 0. SW

‘IrP

419

SEQUENCES

c-11 271-219

C-18 260-283

0:2 0.2 1.5(2)

c-19 266-296

c-9 132-136

O.?(l) 0.2 0.1 LO(l) 1.0(l)

LO(l)

1.0(l) 0.1

2.0(l) O.?(l) 0.2 1.1(11

i::

0.1 0.2 1.0(l) 1.0(l)

1.1(l) Lo(l)

LOU)

O.s(l) 2.0(21 2.0(2) LO(l)

0.3 :::(I,

1.0(l)

0.9(l) 1. I(1)

0.90)

1.1(l)

1.0(l)

0.90)

0.2

1.1(l)

1.9(2) 1.00)

0.9(l)

1.1(l)

1.1(l)

1.00)

2.0(2)

l.OW

k;(l)

0.2

0.3 2.0(Z) 1.10) 0.2 LO(l)

l.O(ll

2.1(2) 1.2(l)

3.0(3)

0.3

C-26 364-369

1.3(l)

2.0(2)

2.0(Z)

1.1(l) 0.2 0.8(l) 0.7

c-27 409-416

C-28 419-424

C-18 271-278

0.2 0.2 0.3

2.0(2)

0.9(l)

1.9(2) 1.00)

C-29 425-431

0.2

0.1

0.9(l)

2.4(Z)

c-30 451-468

c-31 482-498

c-32 500~$06

1.2(l)

0.4

1.9(Z)

LO(l)

l.S@,

0.9(l) 1.2(l)

0.2

LO(l)

0.9(l)

1.40)

2.10)

1.0(l)

0.2

3.0(3) 1.1(l) 0.1 1.1(l)

0.2 0.9(l)

1.0(l) 1,2(l)

LOW LO(l)

+ (1)

c-25 366-363

1.0(l)

c-15 286-216

1.0(l) 1.9(Z) 1.9(2)

0.2 1.0(l)

c-24 325-331

c-14 236-244

1.1(l)

I.101

C-23 318-324

c-13 231-235

c-12 220-230

1.00) 0.90)

c-22 297-309

0.1

4.0(3)

0.9(l) 0.2

c21 291-300

0.1 1.1(l)

1.00) 1.2

c-11 215-219

0.3

1.00) cl2

C-10 193-191

1.W)

0.2

0.1

c-20 294-296

C-8 113-131

0.9(l) 1.1(l)

0.9(l) 0.9(l)

2.0(2) 2.9(3)

c-1 100-105

0.2

1.1(l)

0.1

0.1

0.1 0.8(l) 0.1

LO(l) 0.8(l)

0.1

l.O(ll 0.80) 1.1(l)

LO(l)

0.1

420

SCHROEDER

ET AL,

l.O(ll 0.4

l.O(li 0.9(l)

l.Ofll

1.1(l)

0.3

0.1

0.8(l)

0.9(l) LO(l) l.O(ll l.?.(l) 1.9(Z) 2.1(Z) 0.3 1.9(Z)

0.90)

1.00) LO(l)

0.3 0.6 1.9(Z) 1.60)

1.10)

2.m 1.0(l)

1.0(l) LO(l) LO(l) ,.5(Z)

1.0(l)

3.8(4)

1.W) l.Ofll

LO(l)

2.9(3) 0. ?(I) 0.1

0.1 0.6(l) 1.1(l) 0.1 0.2 0.6(l) 1.W) 0.3 0.3

0.4 2.2(2,

0.80) 2.0(Z) 1.0(l)

0.4 2.0(Z) LO(l) LO(l) 1.1(l)

1.1(l) 1. i(1) LO(l) 1.00) a. O(2) 0.9(l)

1.2fll

1.0(l)

BOVINE

CATALASE

421

SEQUENCES

”“. , 0. I 1. “‘1,

0.3 0.1 ,.“,li

0.9’11 Ll’li

2.8,JI

I.“,*1

0.z

0.5

;::I:; 1.9’21 1.00,

1.0’1,

0.2 *.“,*I

0.2 0.2 0.1

1.0’11

0. I

I.‘421 0.‘

3.“,1)