Amino acid sequences of tryptic peptides of cytochromes b5 from microsomes of human, monkey, porcine, and chicken liver.

Abstract Liver microsomal apocytochromes b5 from man, monkey (Alouatta fusca), pig, and chicken were subjected to trypsin digestion, and all of the peptides were isolated and characterized. The sum of the residues present in these peptides equaled the total amino acid composition of the corresponding parent cytochrome. For all of these peptides a homologous segment was found in our previously established bovine and rabbit cytochrome sequence. This information provided sufficient evidence to construct a unique amino acid sequence for human, monkey, porcine, and chicken cytochrome b5. Comparison of the six sequences indicated a close similarity between these proteins. The human cytochrome differed from that of chicken in 15 positions but from that of monkey in only 2 positions. With the exception of the avian protein sequence, the amino acid replacements were confined predominantly to the NH2- and COOH-terminal segments of the cytochrome. A segment comprising residues 42 to 72 was invariant in all six cytochromes.

OZOLS I From the Department of Biochemistry, University of Connecticut Health Center, Farmington, Connecticut 06036 SUMMARY Liver microsomal apocytochromes & from man, monkey (AZouatfa ~usca), pig, and chicken were subjected to trypsin digestion, and all of the peptides were isolated and characterized.
The sum of the residues present in these peptides equaled the total amino acid composition of the corresponding parent cytochrome.
For all of these peptides a homologous segment was found in our previously established bovine and rabbit cytochrome sequence. This information provided sufficient evidence to construct a unique amino acid sequence for human, monkey, porcine, and chicken cytochrome b5. Comparison of the six sequences indicated a close similarity between these proteins. The human cytochrome differed from that of chicken in 15 positions but from that of monkey in only 2 positions.
With the exception of the avian protein sequence, the amino acid replacements were confined predominantly to the NH2-and COOH-terminal segments of the cytochrome.
A segment comprising residues 42 to 72 was invariant in all six cytochromes.
One intriguing aspect of heme protein structures is that they represent a system in which particular amino acid arrangements of the peptide chain can induce the heme to participate in diverse catalytic functions. Cytochrome bg is a heme protein present in especially high amount in the endoplasmic reticulum of mammalian liver cells (1). Moreover, Halloway and Wakil (2) have shown that cytochrome bg is an integral part of the stearyl coenzyme A desaturase system. Information about the function and, in particular, the primary structure of heme proteins has been accumulating at a remarkable rate during the past few years.
In the last decade, the * A preliminary report of this work has appeared (Fed. Proc., 29, 3794 (1970) amino acid sequences of cytochromes c from about 36 different organisms have been determined, and complete or partial primary structures of hemoglobins of some 20 species are now known (3). The implications derived from these sequences have firmly established the significance of the sequence data in the elucidation of structure and evolution of these proteins. Liver microsomal cytochrome bs has properties that resemble those of the cytochromes c, and also those of the hemoglobins. Thus, in several aspects, cytochrome bs is a member of both the cytochrome c and the hemoglobin group.
The heme structure and ligand-binding properties of cytochrome bg parallel those of the hemoglobins (4). The enzymic and spectral properties of cytochrome bg, however, are strongly related to those of the cytochrome c. An exclusive property of cytochrome bg is its strong association in viva with the cellular membrane components (1).
In previous studies, we determined the covalent structures of calf (5)(6)(7) and rabbit (8) liver microsomal cytochromes bg and briefly discussed the unexpected sequence similarity between this cytochrome and hemoglobin (4). It was also observed that in the rabbit protein heterogeneity at residues 10 and 95 was present (8). In order to explain these findings and to elucidate further the phylogenetic and the structural aspects of this group of proteins, we determined the amino acid sequences of cytochrome bg from man, monkey, pig, and chicken. Independent studies by Tsugita et al. (9) have also established the rabbit cytochrome bg sequence.
For the first 90 residues, these amino acid sequences are essentially the same.
EXPERIMENTAL PROCEDURE Materials and Methods-Microsomal liver cytochromes were obtained as previously described by Nobrega et al. (lo), and, following lyophylization, were stored in glass ampoules in vucuo. The purity of the material was ascertained by the following criteria: the preparations migrated as a single band on DEAE-Sephadex A-50 columns, and when electrophoresed on the cellulose acetate strips in phosphate or acetate buffers (11). The cytochromes used had an absorbance ratio, AS56 reduced to A 280 oxidized, of 1.4 or higher and were homogeneous by end group analysis using the dansyll chloride method (12). Issue of March 25, 1971 F. G. N6brega and J. 0x01s Enzymic Hydrolysis and Isolation of PeptidesHeme-free apocytochrome (0.1 to 0.5 pmole) was prepared by treatment of the cytochrome with acid acetone at 0" as follows: to a 0.5-ml solution of salt-free cytochrome bs in water, 10 ml of cold acetone containing 0.2% HCl (v/v) were added. After 10 min at 5", white precipitated apocytochrome was collected by centrifugation, rapidly dried with a stream of nitrogen, and dissolved in a total volume of 0.5 ml of 0.1 M NHdHCOs, pH 8.1. A 0.1% solution of trypsin (Worthington, n-1-tosylamido-2-phenylethyl chloromethyl ketone-treated) in 0.001 N HCl was prepared immediately prior to use and added to the apoprotein to give an enzyme to protein ratio of 1: 60. After 3 hours at 25", the hydrolysate was lyophilized and dissolved in the buffer appropriate to the desired chromatographic procedure.
The chicken apoprotein was dissolved in 0.1 M Tris-8 M urea buffer, pH 8.1, and the protein solution was diluted 3 times before trypsin addition to reduce the urea concentration to 2 M. After 1 hour at 25", another aliquot of trypsin was added, and the hydrolysis was allowed to proceed for another 5 hours. Peptides were digested with chymotrypsin (Worthington, crystallized three times) in 0.1 M ammonium bicarbonate, pH 8.1, at 25" for 3 hours, with an enzyme to substrate ratio of 1:lOO. Porcine Peptide T-l was digested with pepsin (Worthington, crystallized three times), 0.2 pmole of peptide, 2% (moles per mole) enzyme, 1.5 ml of 0.01 N HCl, 25", 13 hours.
Peptide fractionations by gel filtrations were performed on columns, 0.9 x 60 cm, of Sephadex G-25, equilibrated and eluted with 30% acetic acid. Peptide fractions were identified by the ninhydrin reaction after alkaline hydrolysis (5) and characterized by amino acid analysis.
Tryptic peptides are assigned the letter T-and the peptic peptides the letter P-. The number assigned to these peptides corresponds to the nomenclature adopted previously (6). The number assigned to a residue in these sequences corresponds to the number of the homologous residue of the rabbit protein sequence (8).
Amino Acid Analyses of Proteins and Peptides-Amino acid compositions were determined on acid hydrolysates with the Spinco model 120C amino acid analyzer, equipped with a 6.6-mm flow cuvette and a 4-to 5-mv range recorder, permitting the determination of amino acids in the range of 0.0025 to 0.03 pmole.
When it was necessary to use quantities at the lower limit, The identity of the NH2terminal residue was determined by amino acid analysis on a fraction of the residual peptide.
The number of micromoles of amino acid present in the residual peptide was converted to a molar ratio, by dividing the analytical value of the residue by the mean value of the total composition.
The mean value of the composition was obtained by dividing the sum of the analytical values of all amino acids by the assumed number of residues in the peptide.
The assumed number of residues, if not evident, is given in parentheses following the molar ratio values.
Amino acids present in peptides, before Edman degradations, in yields below 15'% of a residue are not reported or included in the calculations.
The residue marked in boldface type corresponds to the residue removed at each step. A 0.0 molar ratio, following an Edman degradation step, represents less than 0.1 residue. When Edman degradation results are reported in a column form, the column heading C represents the initial peptide composition, and E-followed by a number denotes the composition after the corresponding degradation step. Direct identification and differentiation of the phenylthiohydantoins of glutamic and aspartic acids and their amides was carried out by thin layer chromatography, with solvent C as recommended by Edman and Sjoquist (15). Eastman precoated silica gel sheets were used without fluorescent indicator, and spots were located by spraying with 0.1 N iodine, 5% (v/v) sodium azide solution. Conversion of the phenylthiohydantoin to free amino acid was performed by acid hydrolysis with 6 N HCl at 150", for 16 hours.
The NH%-terminal residues of the proteins were determined after reaction with dansyl chloride according to the method of Gray and Hartley (12). The derivatives were identified by twodimensional thin layer chromatography and polyamide layers by solvent 1 (200 ml of water and 3 ml of 90% formic acid) a,nd solvent 3 (60 ml of n-heptane, 60 ml of n-butyl alcohol, and 20 ml of glacial acetic acid) of Woods and Wang (16).

Amino Acid Composition and Terminal
Residues of Cytochromes -The cytochromes investigated represent the predominant fraction obtained from the final purification procedure by chromatography on DEAE-Sephadex A-50 (11). These fractions were homogeneous as indicated by electrophoresis and spectral ratios of oxidized and reduced states.
The composition of the cytochromes are listed in Tables I through IV. The number of residues of each amino acid in the protein is calculated on the basis of 1 mole of heme per mole of protein.
The human and the monkey proteins each contains a total of 87 residues.
The porcine protein contains 82 residues, and the  Amino acid composition of tryptic peptides of human apocytochrome bs The results are expressed as molar ratios of the constituent amino acids and, except where noted, were obtained by analysis of 20-hour hydrolysates.
No corrections for the destruction of serine and threonine during hydrolysis are incorporated. were separated on a Dowex 1 column giving the elution profile shown in Fig. 1C. Peptides T-3 and T-6 were not separated by this procedure but were resolved by gel filtration on Sephadex G-25 in 30% acetic acid.
Gel filtration of this precipitate on Sephadex G-25 in 30% acetic acid or chromatography on a Dowex 50 column recovered Peptide T-12 in good yield.
The amino acid analyses of the tryptic peptides of human cytochrome bb are given in Table I. The sum of the compositions of tryptic peptides agrees well with the analyses of the parent cytochrome.
The yields represent the actual amount of the peptide with the reported composition obtained. The order of the tryptic peptides of the cytochromes is based on our previously determined calf and rabbit protein sequences as the model.
Since rabbit cytochrome has the longest amino acid sequence, its residue numerology is used throughout this study.
To minimize the volume of repeating data, peptides having composition and properties unamibiguously identical with those of calf and rabbit proteins are listed without proof.
Peptide T-10 (Residues 4 through 9): Ser-Asp-Glu-Ala-Val-&s--Four steps of Edman degradation established the sequence as follows: The two remaining residues were positioned as Val-Lys from tryptic specificity.
Peptide T-10 represents the NHz-terminal region of the human cytochrome b6 because its sequence is homologous only to this region of the protein, in both the calf and rabbit cytochromes. Furthermore, the NHz-terminal residues of the parent protein and Peptide T-10 coincided.
The remaining residues were positioned by homology to the Peptide T-7 from pig cytochrome bg. The results are expressed as molar ratios of the constituent amino acids and, except where noted, were obtained by analysis of 20-hour hydrolysates.
No corrections for the destruction of serine and threonine during hydrolysis are incorporated. The phenylthiohydantoin from the second step of the Edman degradation was identified as the asparagine derivative. The seryl-lysyl sequence was deduced from tryptic specificity.
The low recoveries of residual peptides in the second and third steps were attributed to their slight transfer to benzene and ethyl acetate phases during the extraction steps. The remaining residues were tentatively aligned by homology with the completely sequenced Peptide T-12 from the pig and rabbit proteins.
Peptide T-6 (Residues 33 through %)-The composition and 2 Value from sample hydrolyzed in the presence of 3% thioglycollie acid. the chromatographic behavior ( Fig. 1) of this peptide were identical with that of Peptide T-6 from pig and rabbit proteins. Peptide T-2 (Residues 39 through 51)-This peptide had composition and mobility ( Fig. 1) identical to that of Peptide T-2 from the rabbit cytochrome (8). Thus, it corresponds to the segment consisting of Residues 39 through 51.
Gel filtration of this hydrolysate on Sephadex G-25, as described under "Experimental Procedure" gave three peaks. The first peak consisted of the hexapeptide (Ile, 0.99; Gly, 0.92; Glu, 1.12; Leu, 1.14; His, 1.05; Pro, l.OS), representing the expected NH&erminal segment of the parent peptide. The second peak, a tripeptide, contained the lysyl residue as well as proline and arginine, indicating that it represented the carboxylterminal fragment of T-3, while the third peak was only free aspartic acid.
One Edman degradation step on the tripeptide indicated that lysine was amino-terminal. The data were as follows: Composition: Lys, 1.00; Pro, 1.03; Arg, 0.89.
From the trypsin specificity it was concluded that arginine is carboxyl-terminal and is preceded by proline; hence the sequence of this peptide is Lys-Pro-Arg. Since no other peptides were obtained from the tryptic digests of human apocytochrome bs, and since for all of the peptides described in this section a homologous segment was found in the rabbit or calf cytochrome sequence, the amino acid sequence of human cytochrome bs can be postulated as shown in Figure 5. The results of cyanogen bromide cleavage of human cytochrome bs will be presented in a separate communication.  (Table II) was identical with that of the human protein, except for the absence of the characteristic replacement of the single residue of methionine for leucine, and replacement of 1 tyrosyl residue by phenylalanine.
Comparison of the tryptic digest elution profiles of these two proteins (Fig. 1, C and B) indicates that the noted composition difference is localized to the sequence region comprised by Peptides T-3 and T-4. Table  II summarizes the composition and yields of each peptide isolated from the tryptic digest of the monkey apocytochrome and confirms the above proposed differences between the human and monkey proteins.
Characteristic of Peptide T-3 from all other apocytochromes (6,8) containing isoleucyl-isoleucyl sequence, the isoleucine content of monkey Peptide T-3 increases to 2 residues per molecule only after a 72.hour hydrolysis. Hence, duplicate amino acid analyses were performed only on the initial peptide, and the residual peptide from the last Edman degradation step. Because the chromatographic mobility ( Fig. 1) and the composition (Table II) of all the other tryptic peptides of monkey apocytochromes were identical with those of human or rabbit proteins, and since the sums of the amino acid composition of its tryptic peptides were in good agreement with the over-all composition of the parent protein, the complete primary structure of monkey cytochrome bs must be as shown in Fig. 5.

Isolation and Characterization of Tryptic Peptides of Porcine
Cytochrome &--The elution pattern of the tryptic peptides from a Dowex 1 column is shown in Fig. 1A. This procedure provided peptides accounting for 68 of the 82 amino acids in the porcine cytochrome bs. Similar to the previously investigated apoprotein digests from other species, the nonapeptide (T-12) from the pig protein digest is insoluble in the basic Dowex 1 column elution buffer, and the precipitated peptide may be purified by gel filtration on Sephadex G-25 or may be obtained by Dowex 50 chromatography of the tryptic digest (Fig. 2). As with the calf protein (5), the basic pentapeptide (T-5) is eluted from the Dowex 1 column in a very low yield.
The most satisfactory procedure for the isolation of Peptide T-5 is shown in Fig. 2. Thus, for a complete resolution of all the tryptic peptides of the porcine cytochrome bs, the tryptic digest has to be chromatographed on Dowex 1 and Dowex 50 columns concomitantly. The compositions of the tryptic peptides and the yields in which they were isolated are presented in Table III.
The sum of the ammo acid composition of these peptides accounts for all of the 82 residues of the porcine cytochrome bb. The amino acid sequence of these peptides and their alignment in the parent molecule could be inferred from obvious homologies in composition between these peptides and the tryptic peptides of the calf and rabbit cytochromes.
Nevertheless, all of the porcine tryptic peptides were characterized by the conventional procedures. Peptide T-8 (Residues 7 through 9): Ala-Val-Lys-One Edman degradation step and the tryptic specificity established the sequence of this tripeptide as shown above. The ascending fractions of the Peptide T-8 peak (Fig. 1A)   While the results after the first step show the presence of some aspartic acid and serine, a clear cut decrease of alanine is indicated.
The remaining 2 residues were positioned from the tryptic specificity. Identification of the phenylhydantoins from the second and third Edman degradation steps established that all aspartyl residues were amidated.
It is noteworthy that, despite the presence of tryptophan at residue 3, this peptide underwent six Edman degradations without difficulty. The carboxyl-terminal sequence was postulated to be His-His-Lys from the specificity of trypsin.
Peptide T-6 (Residues 33 through 38): Val-Tyr-Asp-(Leu, Thr)-&s---The application of three steps of the Edman degradation gave results consistent with the sequence proposed from the composition data.
Moreover, their electrophoretic mobilities and chromatographic behavior, in the Dowex 1 system, were identical; hence their sequence must be identical and as postulated above.
To test the validity of this assumption, Peptide T-2 was subjected to Edman degradation.
In the sixth step the anticipated decrease of aspartic acid content did not take place, and after the seventh stage, the composition of the residual peptide was still identical with that of Stage 5 (cf. Table IX).
This suggested that the aspartyl residue at Position 6 was amidated and had undergone cyclization, or that isomerization of this residue to the 0 peptide form had taken place during the Edman degradations. When Peptide T-l was subjected to pepsin hydrolysis, three peptides were formed: P-l, -2, and -3. These were separated by chromatography on Dowex 50 as described in the legend of Fig. 2, except that the column was developed under a linear gradient established between 400 ml of pH 3.1 buffer and 400 ml of pH 5.6 buffer.
Peptide P-l was present in Fractions 29 to 30 (58 to 60 effluent ml).
It was a pentapeptide and had a composition identical with that of the NHt-terminal segment of the parent peptide.
A complete aminopeptidase hydrolysis of Peptide P-l did not confirm that aspartic acid was amidated.
Peptide P-2 8 The results of the Edman degradations on which the amino acid sequences of Peptides T-7, T-5, T-12, T-2, T-l, and T-3 were based (Tables V to X) Table IX. Hence the complete sequence of Peptide T-l is determined as written. Peptide T-4 (Residues 73 through 76): Glu-Leu-Ser-LysThe amino acid analysis of Peptide T-4 fraction was in accord with the proposed sequence except that it contained 0.34 residue of aspartic acid and 0.19 residue of histidine (cJ Table III). The results obtained from two steps of Edman degradation were consistent with the proposed sequence.
Peptide T-J (Residues 77 through 88): Thr-Phe-Ile-Ib-Gly-Glu-Leu-His-Pro-Asp-Asp-Arg (Table Xa)-Edman degradations established the sequence of the first nine amino acids. Hydrolysis of Peptide T-3 with 0.03 M HCl at 107" for 14 hours released 2 eq of aspartic acid and 1 eq of arginine.
Since no other amino acids were released, the COOH-terminal sequence must, therefore, have the sequence as written.
The foregoing results lead to the primary structure for porcine cytochrome bs shown in Fig. 5 The characteristic turbidity which appears a few minutes after the addition of trypsin failed to disappear during the usual incubation period. Fractionation of this digest on Dowex 1 column afforded low yields of all peptides.
Hence, apocytochrome was denatured in 8 M urea, prior to the tryptic hydrolysis, and the digestion was performed in 2 M urea. After 5 hours at ambient temperature the hydrolysate was essentially clear. Fig. 3 shows the elution pattern of this digest from a Sephadex G-25 column.
Amino acid analysis of Peak 1 fractions suggested that it is a mixture of T-l, -2, -3 and -12. When N-ethylmorpholine-picoline-pyridine acetic acid buffer, pH 9.4 was added to the lyophilized fractions of Peak 1 a slightly turbid solution was obtained. The insoluble material was Peptide T-12, confirmed by chromatography on a Dowex 50 column (cf. Fig. 2). Chromatography of the soluble portion on a Dowex 1 column afforded Peptides T-l, -2, and -3 (Fig. 4A). Peptides comprising Peaks 2 and 3 of Fig. 3 were pooled and separated by chromatography over Dowex 1 as shown in Fig. 4B. Four peptides were found: T-8, -7A, -6, and -7B.
The compositions of these peptides and the yields in which they were isolated are presented in Table IV.
The sum of the amino acid compositions of these peptides is in good agreement with the amino acid composition of the parent protein.
Since Peptide T-8 is the only peptide with glycine at the NH2 terminus, it must be the NH2terminal peptide of the original protein.
Peptide T-7A (Residues 10 through 12): Tyr-Tyr-Arg-Two steps of Edman degradation removed all the tyrosine; therefore, the sequence of Peptide T-7A must be as written above.
Step 1  with 30% acetic acid. After 10 ml, l-ml fractions were collected at a flow rate of 7 ml per hour. must be homologous to the 3 NHz-terminal residues of porcine Peptide T-7.
Peptide T-7B (Residues 13 through 18) : Leu-Gln-Glx-Val-Glx-Lys-Four steps of Edman degradation determined its sequence which identified it as the homologous COOH-terminal segment of Peptide T-7.
The data were as follows. 2.00 residues of aspartic acid, and 0.15 residue of serine.
Since no other amino acids were released, these results are consistent with the proposed NHzterminal His-Asp-Asp-Ser sequence. When Peptide T-12 was subjected to chymotryptic hydrolysis and the resulting digest was fractionated by gel filtration on Sephadex G-25 (cf. Fig. 3), two peptide-containing peaks were found.  Fig. 3) on a Dowex 1 column (60 X 0.9 cm). Elution was begun with a mixing chamber and reservoir containing pH 6.3 buffer. At the fractions indicated by the arrows on the elution diagram, the buffer in the reservoir was replaced by the buffer indicated..
Fractions of 1.7 ml were collected at a flow rate of 27 ml aer hour. and 100-J aliquots were analyzed by the ninhydrin method. A, resolutron of Peak 1; B, that of Peak 3.
acid composition that was identical to the parent peptide. It was recovered in 50% yield. The incomplete chymotryptic cleavage of Peptide T-12 can be partly attributed to the low solubility of this peptide at neutral pH. The composition of the second peak (Fractions 24 and 25) again was identical with that of Peptide T-12, but the results of Edman degradations indicated that these fractions consisted of two peptides in about equal proportions.
The anticipated chymotryptic cleavage at the tryptophanyl residue afforded these two peptides. Three stages of phenylisothiocyanate degradation were applied to this peptide mixture.
Peptide T-6: From trypsin specificity, the carboxyl-terminal sequence must be Thr-Lys.
Peptide T-2 (Residues 39 through 61): Phe-Leu-Asp-Glu-His-Pro-Gly-(Gly , Glu, Glu, Val, Leu)-Arg-The composition of the avian Peptide T-2 indicated that it differs from Peptide T-2 of other species by having 1 additional residue of aspartic acid and 1 less residue of glutamic acid. Edman degradation indicated that this interchange is at residue 41 and confirmed that the sequence of all the other residues must be identical with that of porcine Peptide T-2.
Peptide T-S (Residues 73 through 90): Ala-Leu-Ser-Glu-Thr-Phe-(Ile , Ile, Gly , Glu, Leu, His, Pro, Asp, Asp) -Lys-Pro-Arg-The composition of this peptide indicated that it is homologous to the segment in the porcine protein represented by Peptides T-4 and T-3.
Fractionation of this hydrolysate on a Sephadex G-25 column (cf. Fig. 3 The residues which are different from the rabbit protein are indicated above the continuous sequence. a From Ozols and Strittmatter (6,7); b 0~01s (8) Direct examination of an aliquot of the native tripeptide on the amino acid analyzer revealed only a peak emerging at the position just before lysine; hence the nature of the unknown ninhydrin positive component, present in the dilute acid hydrolysate of Peptide T-3, is established. These results support the sequence of Peptide T-3 as written above, and the complete amino acid sequence of the chicken cytochrome bs must be as shown in Fig. 5.

DISCUSSION
The complete amino acid sequences of the human, monkey, porcine, and chicken cytochromes bg are deduced from the results of the tryptic peptide characterization. The total number of residues derived from the compositions of these peptides, listed in Tables I to IV, are in good agreement with the compositions of the parent proteins.
The similarities between the tryptic peptides sequenced in this study and those from calf (6,7) and rabbit (8) cytochromes readily permit alignment of the peptides in a unique continuous order, as delineated in Fig. 5. The sequence of two tryptic fragments from the avian cytochrome further strengthens the assumption that the order of tryptic peptides is identical for all the cytochromes 65. The absence of trypsit-sensitive bonds in the avian protein segments comprised of Peptides (T-5)- (T-12) and (T-4)-(T-3) confirms that they are linked in that order.
The deduced linear structures are also in accord with the studies on the NH&arminal residues of the parent proteins (10). In view of the above findings, and since tryptic hydrolysis did not yield any unanticipated fragments from these cytochromes, characterization of the chymotryptic peptides was not necessary in order to confirm the assigned tryptic peptide order.
An important point of the peptide separations methodology merits discussion: of great value as a means of separating tryptic peptide mixtures of cytochromes bb is the Dowex 1 chromatography, using buffers developed by Rudloff and Braunitzer (18) and Schroeder and Robberson (19 resins. Asalready noted elsewhere (7), this factor led to a gap in the initially proposed calf cytochrome bs sequence. Another peptide segment in which fractionation difficulties are encountered is Peptide T-5.
Peptide T-5 with the sequence His-Asn-Asn-Ser-Lys cannot be identified in the Dowex 1 column eluate, whereas the His-Asn-His-Ser-Lys peptide is recovered in satisfactory yields (cf. Fig. 1). Nevertheless, the consecutive use of Dowex 1 and Dowex 50 columns together with the use of an amino acid analyzer with sensitivity in the 0.005 pmole range proved to be a very satisfactory means for quantitative recovery and characterization of peptides derived by tryptic hydrolysis of limited quantities of cytochrome 6s. Indeed, the foregoing work was accomplished with 0.4 pmole of the human, pig, and chicken protein, and 0.1 pmole of the monkey cytochrome bg.
Of the four cytochrome bs species investigated in this study, tryptic cleavage of three species proceeded in the anticipated fashion.
Tryptic hydrolysis of the chicken apocytochrome proceeded in an unexpected manner. Even after more trypsin was added and the incubation time was prolonged, fractionation of this digest afforded a very low yield of all peptides.
A complete hydrolysis of the avian apoproteins was achieved by denaturing the apocytochrome with urea prior to hydrolysis and performing the reaction in 2 M urea. It should be mentioned that the chicken protein has essentially the same number of trypsinsusceptible sites as the other proteins.
Indeed, the avian protein has 1 less adjacent acidic residue, at arginyl residue 72, which may even be expected to increase the rate of Peptide T-l accumulation.
Peptide T-l, however, was not found in appreciable yield in this hydrolysate.
Such behavior is in contrast to that of the rapidly occurring tryptic hydrolysis at arginyl residues 51 and 72 of the acetylated apocytochrome.
A satisfactory in- Comparison of the primary structures of cytochromes 66 from five mammalian and one avian species reveals that in the common region (residues 4 to 90), 24 residues are variable and 60 are invariant.
Interestingly, the amino acid replacements, particularly among the mammalian cytochromes, are confined to the terminal segments of the protein.
On the whole, the extent of the residue variance in these sequences is in accord with the zoological classification. Human cytochrome differs from that of monkey (Alouattu fusca) by 2 residues, and from that of chicken by 15 residues.
Of the 15 variant residues between the pig and the chicken proteins, 6 are basic residues, yet there is a remarkable conservation of the net charge of the protein.
Indeed, the chicken and the pig cytochromes have an identical electrophoretic mobility (10). An examination of the amino acid replacements in terms of the nucleotide interchange for the codon of each amino acid (Table XI) reveals that all variations involve a single base change.
The three loci involving two-base changes are among the mammalian cytochrome sequences. Although there appears to be a preponderance of mutation pathways involving adenine to guanine fluctuations, in total there are equal numbers of transitions (purine/purine, or pyrimidine/ pyrimidine) as of transversion (pyrimidine/purine) mutations. Because of some missing terminal fragments, presumably containing variable residues, a comparison of the rates of evolutionary variations of cytochromes bg and other heme proteins is premature.
Nevertheless, the following statement may be made. The variance of the cytochrome bg sequence is by no means unique, when compared with that of other heme protein sets, nor is it identical with that of hemoglobins or cytochromes c, but agrees with the conclusion drawn from such studies, namely, that proteins undergo sequence variation in the course of evolution at very different rates (3). Do the terminal segments in cytochromes bg, in general, undergo mutations at a statistically significantly greater rate than that of other regions? Evidence on this question must wait until more sequences from different species are obtained.
In respect to the continuous invariant segment (residues 42 to 72), it is tempting to speculate that it is invariant because the functional conformation of the protein is critically dependent on that particular amino acid arrangement. Indeed, the two imidazole groups, spaced 24 residues apart (residues 43 and 67) certainly meet the requirements to represent the heme-binding site of this cytochrome.
Hence, mutations in this region may be lethal not only to the heme binding, but also to the normal intracellular function and turnover of the protein.
Such an argument, however, would also imply that mutations in the apparently variant NH2 and COOH terminal segments do not result in a significant structure alteration. And then it stands to reason that a single species should also contain a population of cytochromes having sequences that vary in these regions. Consistent with such reasoning is the finding that rabbit cytochrome bg is heterogeneous at residues 10 and 95 (8). In the present studies, however, no evidence of polymorphism was observed.
It may indeed be possible that polymorphism of a small degree could have escaped detection, since only a small quantity of cytochromes were examined in this study.
The results of current studies on the sequence of cytochrome bs components of an individual human liver, perhaps, will be more decisive.