Specific covalent labeling of cytochrome P-450CAM with 1-(4-azidophenyl)imidazole, an inhibitor-derived photoaffinity probe for P-450 heme proteins.

A generally applicable photoaffinity labeling procedure for the active site of P-450 heme proteins has been developed using 1-(4-azidophenyl)imidazole (API), a photolabile analogue of the common inhibitor N-phenylimidazole. The binding of API to P-450~~ (& 1 PM) elicited a type II spectral shift of the Soret band. Irradiation of the protein l ligand complex at 313 nm caused specific covalent binding. Under similar conditions affinity labeling of pancreatic ribonuclease was negligible. Reconstitution experiments demonstrated that API attachment was accompanied by inhibition of camphor hydroxylation. At cyctochrome P-450~~~ concentrations up to 10 gM and API concentrations up to a g-fold molar excess of label over protein, covalent binding increased linearly with label concentration until saturation of a single site was reached at 114% incorporation of tritiated label. More concentrated protein solutions (40 to 50 PM), however, engendered gradual specific labeling at a second discrete site. In these experiments covalent incorporation of API substantially in excess of 100% was observed. The monoand dilabeled protein derivatives were readily resolved by gel electrofocusing (~15.2 and 5.8). A small heme-bearing fragment was isolated by Sephadex G-75 chromatography (Mr 5.0 to 5.5 x 103) from API-labeled cytochrome P-450~~~ after cyanogen bromide degradation. This heme peptide fraction contained -80% of the heme of cytochrome P-450~~~ and -70% of the incorporated label; its Soret maximum was at 356 nm in contrast to dissociated heme (390 nm). No significant amount of heme or label was associated with any other BrCN fragment.. The resolved monoand dilabeled heme peptides (~14.2 and 4.8) contained all radiolabel associated with the heme peptide fraction. Thus, photocovalent API labeling of cytochrome P450CAM occurred almost exclusively in the close vicinity of the heme. The binding sites of cytochrome P-450~~ for the heme group and both labels are encompassed in

A generally applicable photoaffinity labeling procedure for the active site of P-450 heme proteins has been developed using 1-(4-azidophenyl)imidazole (API), a photolabile analogue of the common inhibitor N-phenylimidazole.
The binding of API to P-450~~ To whom correspondence concerning this paper should be addressed. a tightly structured domain which can be released in the form of a small heme peptide by selective chemical cleavage. Attempts to understand the mechanism of monooxygenase reactions have focused on the interaction of the terminal oxidase component, cytochrome P-450, with dioxygen, substrates, or inhibitors. Binding of substrates or inhibitors to P-450 heme proteins has received special attention because it is usually accompanied by a pronounced spectral shift of the Soret maximum suggesting a conformational change in the heme ligand arrangement (1,2). Depending on their direction to lower or higher wavelengths, these have been classified into essentially three groups known as type I, associated with a low to high spin conversion of the heme iron, type II, and modified type II (equivalent to reversed type I) spectral changes (3, 4). But compounds eliciting a similar type of spectral response in liver microsomal P-450 heme proteins may have quite unrelated chemical structures. Even with cytochrome P-450~~~ of the bacterial camphor hydroxylase (5) which shows much higher substrate specificity than the microsomal P-450 heme proteins, it is difficult to rationalize the spectral responses elicited by ligand binding. For instance, binding of the substrate, n-(+)-camphor, causes a strong type I shift while binding of the product, hydroxycamphor, occurs without concomitant spectral change (6). Moreover, chemical modifications of certain hydrophilic amino acid side chains by alkylation can cause shifts reminiscent of substrate binding (7).
Among the more promising current efforts to scrutinize the basic effect leading to the spectral manifestations are kinetic and static studies at subzero temperatures (8), correlations of the spin state of the P-450 heme iron with the electronic configuration of the ligand molecule (9), and studies in model systems (10,11). In the absence of data on the chemical composition and the exact location of the binding site for a given substrate or inhibitor eliciting a pronounced spectral shift, however, it would appear that our understanding of the nature and size of the effect will remain quite limited because the heme and substrate binding domain may be expected to employ unique structural features for this purpose. At present there is no assurance that a distinct spectral shift upon binding really implies closeness of the ligand to the heme.
The present communication describes synthesis and application of 1-(4-azidophenyl)imidazole a simple, photolabile derivative of the general P-450 inhibitor, N-phenylimidazole, which due to minimal structural alterations still binds with high affinity and specificity to several P-450 heme proteins which have been available for testing ' (12, 13). This reagent Photoaffinity Labeling should be applicable to photoaffrnity labeling of a large number of functionally different P-450 heme proteins . Thus use of  this reagent to determine the location and chemical nature of  the inhibitor  binding site by means of photocovalent  attachment should open a new avenue for comparative  structural  studies of P-450 ligand binding sites.  This paper outlines the rationale of the photocovalent  approach to active site studies and gives the details of a simple  photoaffinity labeling procedure applied to P-450cA~' of the camphor 5-exo-methylene hydroxylase of Pseudomonas putida (14, 15) which still is the most specific and best characterized P-450 heme protein available today. Partial accounts of certain aspects of this research have been communicated previously (12,16,17 Unless otherwise stated all experiments with cytochrome P-450cAM were carried out in 50 mM potassium phosphate, pH 7.5, which will be referred to as buffer throughout the text.

Photoaffinity
Labeling of P-450~~ with API Binding Assay-The spectral dissociation constant, KU, for ligand binding to cytochrome P-450~~~ was determined by difference spectroscopy (22, 23) on an Aminco-Chance dual wavelength spectrophotometer at room temperature. In a typical experiment 1.0 ml of a 2.5 PM cytochrome P-450~~~ solution in buffer was placed in each of two l-ml (l-cm path length) quartz cuvettes and a base-line of equal light absorbance was recorded. After each consecutive addition of an ethanolic API solution, and an equal volume of ethanol, to the sample and reference cuvettes, respectively, the difference spectrum was recorded.
A plot of the reciprocal value of the peak to trough absorbance change (AA) as a function of the reciprocal of the API concentration yielded a straight line which was extrapolated to the abscissa to determine the KIJ. Photolysis Apparatus-Photolysis experiments were performed at 313 nm employing the apparatus shown in Fig. 2. A polished aluminum reflecting shield was used to direct the emission of a 450 W Hanovia medium pressure quartz-mercury lamp (Ace Glass, Vineland, N. J.) upon an aluminum cuvette housing placed 7 cm from the lamp. The solution to be photolyzed was placed, for preparative experiments, in a 3-ml stirred quartz cuvette (l-cm path length) located in position 3 ( Fig. 2) (26), the gels were cut into 1.5mm slices and each slice was allowed to stand in a tightly capped (polyethylene liner) 20-ml glass scintillation vial with 0.5 ml of 30% HZ02/concentrated NH,OH (99:l). After the gel slice had dissolved (1 to 3 days at 23°C in the dark), 10 ml of liquid scintillation mixture were added and counting was carried out at 4°C. The conditions applied for PAGIF of the BrCN-derived hemebearing fragment, resolved by Sephadex G-75 chromatography, were the same as described for the heme protein and its API derivatives.

RESULTS
API was synthesized by a 3-step procedure in 28% overall yield from imidazole and p-chloronitrobenzene as outlined in Fig. 1 (Fig. 3, upper inset). The spectral dissociation constant, Ko, obtained from the double reciprocal plot of these data (Fig. 3,  After recording a baseline of equal light absorbance with 1 ml of a 2.5 pM cytochrome P-450~~~ solution in each cuvette, aliquots of an ethanolic solution of API and an equal volume of ethanol were added to the sample and reference cuvette, respectively. A plot of the peak to trough change in optical density (AOD) versus API concentration yields a hyperbolic curve (upper inset) and a double reciprocal plot gives a straight line from which the KI, is obtained by extrapolation to the abscissa (lower inset).
cytochrome P-450~~~ between the aromatic protein bands and the Soret band of the heme group, filters were used to select 313 nm as the compromise wavelength at which API was still readily photolyzed while the heme protein sustained only minor photodamage when maintained at 4°C. In the photolysis apparatus in Fig. 2, photodecomposition of API to the corresponding nitrene (27) ensued upon irradiation with light of 313 nm (details under "Methods") with a first order rate constant of 0.459 h-' (t1,2 = 1.51 h). These kinetic parameters were determined in the absence of cytochrome P-450~~~. The intensity of light available for activation of API is somewhat reduced in a photoaffinity labeling experiment since P-450~~~ at 40 to 300 pM exhibits significant absorption at 313 nm. The first order rate constant obtained in the absence of protein was therefore used only as a rough guideline and the optimal irradiation time for efficient photoaffinity labeling was determined experimentally to be 5 h. Under identical conditions, irradiation of the N-phenylimidazole complex of cytochrome P-450~~~ caused a small increase in absorption in the 200 to 315 nm region roughly equivalent in magnitude to the concomitant decrease in the Soret absorption (data not shown). Photooxidation of selected amino acid residues has been reported to occur through the porphyrin-mediated photosensitization of heme proteins to visible light (28). Direct photodestruction of cysteine, methionine, tyrosine, and tryptophan has also been observed upon irradiation at wavelengths greater than or equal to 280 nm while they are stable to irradiation at wavelengths greater than or equal to 320 nm (29). It should be emphasized, however, that these changes occurred at a rate much slower than the rate of photolysis of API. Because of the favorable ratio of photoactivation of API to photodamage to P-450~~~ upon irradiation at 313 nm, photoaffinity labeling with this heme protein was routinely carried out at this wavelength.
Prior to photolysis unbound substrate was removed from with buffer. For analytical experiments, 3 nmol of heme protein and a g-fold molar excess of API were dissolved in 0.3 ml of buffer and photolyzed at 4°C for 5 h. Separation of cytochrome P-450~~~ from excess API and its unbound decomposition products was carried out on Sephadex G-10 equilibrated with buffer. The results of four typical experiments pertaining to the photolysis conditions and controls are presented in Fig. 4. As shown in Fig. 4A, in the absence of photolysis only a trace of API travelled unresolved from cytochrome P-450~~~. Interestingly, a trace of unresolved API was found after sieve chromatography in all photoaffinity labeling experiments. This trace representing no more than 1 to 5 mol, however, was readily removed from the protein by a second passage through the Sephadex column or by exhaustive dialysis. Thus no covalent attachment had occurred in the absence of photolysis. When P-450~~~ was irradiated in the presence of prephotolyzed API a similar trace of radioactivity was found in the protein peak after sieve chromatography (Fig. 4B), and again it was easily removed by dialysis. On the other hand, Fig. 4C demonstrates that significant amounts of API are covalently associated with the protein when the photolysis product of P-450~~~ with API is chromatographed using the same column procedure. Neither dialysis nor chromatography could further resolve API from the P-450~~~. According to Fig. 40, API covalently labeled pancreatic ribonuclease but only to 3 mol % under conditions identical with those in which cytochrome P-45OcAM was labeled roughly stoichiometrically. Sieve chromatography on a column of Sephadex G-10 (0.8 cm x 25 cm, 12 ml) equilibrated with buffer was performed on 3 nmol of protein and 27 nmol of [JH]API dissolved in 0.3 ml of buffer after a 5-h incubation period in the dark or after 5-h photolysis at 313 nm. The column effluent was monitored at 280 nm; 100 ~1 of each 0.9-ml fraction were combined with 10 ml of liquid scintillation mixture and counted. A, resolution of P-450~~~ from API without prior photolysis; B, resolution of P-450~~~ from prephotolyzed API after photolysis; C, resolution of API-labeled P-450~~~ from excess API; D, resolution of APIlabeled pancreatic ribonuclease from excess API.
The dependence of covalent labeling of P-450~~~ with API on label concentration is examined in Fig. 5 using a 10 pM protein solution. Photocovalent labeling increased linearly with label concentration until 114% incorporation had occurred. Since increase of label concentration beyond a g-fold molar excess over protein concentration did not cause significant increases in total covalent label attachment, this saturation curve is consistent with the interpretation that essentially a single site was labeled. The amount of label incorporated in excess of 100% may be indicative of the extent of labeling which occurred at other sites. In order to determine how much of the bound label resides at a single location, the API derivatives of P-45OcAM resulting from photolysis at various label concentrations were investigated by gel electrofocusing and by reconstitution experiments. Since the strongly basic group contributed by the label will alter the PI of the protein derivative it can be expected that labeling with API will produce new protein species clearly separable from unlabeled protein due to a difference in PI.
Thus the API derivatives of P-450~~~ were resolved by 120 100 0-l .E ; 80 I /--/. on cytochrome P-450 CAM (3 nmol, 10 pM) and API in 3, 6, 9, or 12-fold molar excess. of P-450~~ with API PAGIF and located on the gel by measuring radioactivity (Fig. 6). Under analytical conditions of photoaffinity labeling (protein concentration = 10 pM) incorporation of API was limited to the high affinity binding site, yielding a mixture of monolabeled and unlabeled protein which can be readily resolved by gel electrofocusing.
The extent of incorporation of photoaffnity label into the second binding site did not exceed 5% at 18fold molar excess of API over protein and was hardly noticeable at lower photoaffinity label concentrations (data not shown).
As shown by the examples given in Fig. 6 (A and B), API incorporations substantially in excess of 100% were, however, observed when e.g. 40 or 100 pM P-450~~~ solutions were photolyzed in the presence of g-fold molar excess of photoaffinity label. Values of p1 5.2 and 5.8 were measured for the mono-and dilabeled API-derivative of P-450c~~, respectively. These results suggest that the second molecule of API incorporated per molecule of P-450~~~ is at a specific binding site rather than resulting from adventitious labeling of several nonspecific sites. If labeling were occurring at several nonspecific sites of approximately equal affinity for API, one might logically expect to observe in PAGIF, a statistically determined population of protein molecules containing 2, 3, and 4, etc. API molecules/molecule of P-45OcAM. No derivatives containing more than 2 molecules of API/molecule of P-450~~~ have been observed. Only three protein bands are present after PAGIF of photolysis mixtures resulting from preparative labeling experiments. The band of unlabeled protein is getting progressively smaller while increased amounts of dilabeled derivative are found with increases in protein and API concentrations beyond the analytical range.
The amount of unlabeled protein was assessed by measuring only absorption at 600 nm.
Reconstitution of camphor hydroxylation with API-labeled P-450~~~ was carried out according to the procedure of Gunsalus and Wagner (15). When prephotolyzed P-450~~~ (5 h in the absence of API) was employed in the reconstitution experiments, a 40% reduction in the rate of camphor hydroxylation was observed. Although these results indicate a photoinactivation of P-450~~~ which is independent of photoaffinity labeling, a further decrease in the rate of hydroxylation, which is attributable to specific covalent labeling, was observed when API-labeled P-450~~~ was employed in the reconstitution. concomitantly and perhaps in competition with each other. It is therefore difficult to determine the exact contribution of each to the final reduction in rate which is observed. When 70% API-labeled P-450~~ was employed in the reconstitution, an 83.4% reduction in the rate of the enzymatic reaction was seen. But this does not necessarily imply that the reduction in rate due to photoinactivation was 13.4% since no linear relationship between the extent of API-labeling and the reduction in hydroxylation rate has been established as yet. Reconstitution with 114% API-labeled P-450~~~ showed that no more than 7.5% of the hydroxylase activity remained.
In order to locate the high affinity binding site of API on the linear polypeptide chain, extensively monolabeled cytochrome P-450~~~ was subjected to BrCN degradation under conditions which lead to limited degradation with the unlabeled protein (30,31). Fig. 7 shows the resolution of the BrCNderived fragments on Sephadex G-75 equilibrated with 20% acetic acid. Although not indicated in the graph, the concentration of peptide material was determined by amino acid analysis of the fractions in parallel to 280 nm absorption to ascertain the concentration of peptides deficient in aromatic residues and to correct for contributions of the photoaffinity label to the absorption in the UV region. While only a rough fractionation of the larger fragments was achieved by this separation step, a low molecular weight fraction containing -80% of the bound heme (Fig. 7A), and eluting immediately preceding the dissociated heme (-14%), was well resolved from the group of heme-free fragments. This heme-associated peptide fraction also co-eluted with -70% of the radioactivity incorporated with the label. Since the absence of undigested protein in this digest reflects the advanced state of selective degradation of the protein, it may be assumed that this amount of radioactivity accounts roughly for the amount of specifically bound API while the remaining 20% of covalently bound API migrating with the larger fragments represents the extent of nonspecific labeling. The heme-associated peptide fraction eluted at an effluent volume corresponding to a molecular weight of 5.0 to 5.5 x 103. As shown in the upper inset in Fig. 7A, gel electrofocusing of this fraction indicated the presence of one radiolabeled BrCN fragment with a pI 4.2 corresponding to the monolabeled heme peptide. Thus, the available evidence suggests that this fragment includes the specific binding site of API.
The same BrCN degradation experiment was also carried out with an API-labeled P-450cA~ preparation which contained 168% of label distributed over mono-and dilabeled derivatives (Fig. 7B). In this case, much more heme had been dissociated (-31%) and the heme peptide fraction contained >88% of the radioactivity but only 55% of the bound heme. This finding indicates progressive heme dissociation with increased labeling at the second site. Gel electrofocusing of the heme peptide fraction demonstrates the presence of two radiolabeled bands suggesting that the second site is also located on the heme peptide since the total counts are now distributed over two bands of the expected p1 values and in proportions corresponding to their distribution in the protein preparation. Fig. 8 compares the Soret band of heme peptide material corresponding to the ascending slope (l), the peak fraction (2), and the descending slope (3) of the heme-associated fraction eluted from Sephadex G-75 (Fig. 7B). The Soret maxima of unlabeled, mono-, and dilabeled heme peptides were all found to be at 356 nm which is distinctly different from the Soret maximum of heme extracted from P-450~~~ with a mixture of acetone and ether after acidification (390 run) and the maximum of the ferric P-450~~~ (417 nm) from which it is derived. Peptide material eluting with the ascending slope of the fraction contains almost exclusively heme peptide, while the center peak region shows the presence of a mixture of heme peptide, dissociated heme, and peptide. Ma-

Photoaffinity
Labeling of P-450~~ with API FIG. 9. Schematic representation of the preparation of API-labeled P-450~~~ heme peptides. terial from the descending slope and the tail of the fraction contains mostly dissociated heme together with a small amount of apopeptide.

DISCUSSION
Our concept of the active site structure of cytochrome P-450cAM where the specific binding of the photoaffinity labels takes place and the subsequent generation of a small heme peptide is schematically summarized in Fig. 9. The scheme proposes probable positions for substrate and label in the microenvironment of the heme and a preferred orientation of the inhibitor-derived label, API, toward the heme group. The position of the putative substrate-binding sulfhydryl group is also indicated (32). The amino acid side chains acting as chelating groups to the heme iron in the coordination positions 5 and 6 are indicated as an imidazole group of histidine and a sulfhydryl group of cysteine (8,11,33,34).
API was readily synthesized (Fig. 1) and radiolabeled; it proved to be a specific high affinity label for the active site of P-450~~~. Because addition of the azido group in para position of the phenyl ring caused only a minimal change in size and shape of the molecule, it can be assumed that this addition did not change the orientation of the ligand relative to the heme group. This is supported by the typical type II spectral shift (Fig. 3) and the favorable spectral dissociation constant of API. From these data it is apparent that API binds in the same fashion as its parent compound and with an affinity that is at least as high as that of camphor, the natural substrate of P-450,~~. In contrast to substrate-derived photoaffinity labels, API has a much wider range of potential applications for P-450 heme proteins regardless of their bacterial, mitochondrial, or microsomal origin. In fact, it has already been shown that it can bind tightly and specifically to the microsomal heme proteins P-450~~-2 and P-450i,M-44 (12) of rabbit liver and to P-450 heme proteins of Rhizobium japonicum (13). Thus, the stage is now set for the comparative structural study of the inhibitor binding site of those P-450 heme proteins which bind API with high affinity.
It is important to realize that the photolysis procedure applied in this study evolved as a compromise solution in response to our needs for specific, covalent, active site directed probes tailored to the unique properties and requirements of P-450 heme proteins. Obviously, photoactivation of API could be carried out more efficiently at wavelengths closer to its absorption maximum at 265 nm but the absorption characteristics of P-450 heme proteins necessitated the selection of 313 nm instead (Fig. 2). Although photoactivation of API proceeds only at a modest rate at this wavelength, it was chosen to avoid significant photodamage to the heme protein which shows strong absorption throughout the region between 200 and 600 nm except for a window of minor absorption between 305 and 315 nm.
Since effective photolysis has to proceed for a time period in excess of several half-lives of the label, the need for extended light exposure of the heme protein was clearly established. But this cannot be done completely without penalty, and a small but spectrally recognizable photoinduced alteration of Photoaffinity Labeling of P-450~~~ with API 7245 the P-450 heme protein was unavoidable. Thus, the conditions the heme and associated substrate or inhibitor binding sites. selected for our photolysis procedure were designed to keep With the introduction of radioactivity a firm balance has been this effect to a minimum while simultaneously adjusting to obtained for the amount of label associated with the small the modest power output of the mercury lamp at 313 nm.
heme peptide as shown in Fig. 7 (70%). Only small amounts of For proof of specific photocovalent attachment of API to radioactivity were found associated with any of the other P-450~~~ the following four conditions were applied: (a) co-BrCN fragments.
From this evidence we conclude that valent binding of label to protein must be shown to result monolabeled P-450 CAM contains roughly a stoichiometric solely from photolytic activation of API, and (b) it must reach amount of API bound to a single high affinity site in the saturation at a level close to stoichiometry; (c) thus only immediate environment of the heme. proteins with high specific affinity for this label can show This conclusion is reinforced by the results from gel electrosignificant covalent attachment, and (d) this attachment must focusing of the API-derivatized heme peptide (Fig. 7, upper occur at a discrete site, preferably in the vicinity of the heme insets) which showed the presence of one and two major group.
peptide bands, respectively, coincident with label. The extent to which our approach was successful in meeting While it is highly probable that the single residues of these conditions is demonstrated in Figs. 4 through 7. From histidine and cysteines found in the heme peptide (31) are Fig. 4 we conclude that no covalent binding of API occurred indeed those involved in heme chelation in the parent protein, in the absence of photolysis and that a protein without specific no convincing evidence can be provided at this point to show API binding sites, such as ribonuclease, will not be photoco-that this assumption is truly justified. Seen from this perspecvalently labeled by the procedure applied in our investigation.
tive the spectral characteristics of the heme peptide are at From Fig. 5 Fig. peptide with a Soret maximum at 356 nm can also be obtained 6. At low protein concentration and g-fold molar excess of from P-450~~~ ,in the absence of covalently linked substrate label essentially only one site is labeled while solutions con-or inhibitor and does not reflect the influence of the label." taining protein and label at higher concentrations promote But in this case it represents a very small fraction of the covalent labeling at a second specific site of lower affinity.
isolated heme peptide while most of the heme peptide prep-Since the objectives of this study focus on complete saturation aration contains heme loosely associated and shows a Soret of the high affinity inhibitor binding site, we have chosen peak at 390 nm which coincides with the maximum exhibited stoichiometric labeling of this site as the reference point to by heme extracted from P-450~~~. Thus it appears that under which the amount of API incorporated is compared. Thus the conditions of BrCN cleavage some of the heme can be 114% API-labeled protein comes very close to having the high dislodged from its original position, and while it is still enaffinity site labeled to 100% while the rest accounts for the trapped in the pouch-like heme peptide domain, it has aslevel of nonspecific labeling. This interpretation is consistent sumed spectral characteristics similar to those of dissociated with the results of our attempts to reconstitute camphor heme. Obviously, the heme ligation of the BrCN-derived heme hydroxylation with the API derivatives of P-450 CAM. A prep-peptide has to be scrutinized by the same physicochemical aration containing 70% label can still show a moderate cata-procedures that were successfully applied to the parent prolytic activity but after 114% of API attachment almost all tein, especially electron spin resonance spectroscopy. catalytic activity is effectively suppressed. From this finding The isoelectric points of the API-labeled heme peptides, p1 it is concluded that API is indeed bound at the active site and 4.2 and 4.8, contrast with that of the unlabeled heme peptide, thus prevents camphor binding and hydroxylation.
It is of interest that reconstitution of camphor hydroxylation with the API-labeled preparations of P-450~~~ also displays the characteristic rate enhancement with increasing amounts of putidaredoxin known to be obtained for the unlabeled protein (35,36).
Digestion of the substrate-protected heme protein with BrCN was previously found to cause only limited degradation of P-450cA~ and to produce a series of heme-bearing fragments p1 4.0, and these values are comparable to p1 4.5 for the unlabeled (33), and ~15.2 and 5.8, for the mono-and dilabeled hemeprotein, respectively. Due to the basic character of API the mono-and dilabeled derivatives exhibit p1 values which are progressively closer to neutrality than that of their acidic parent compounds.
In this context it should be emphasized that gel electrofocusing provides significant advantages over sodium dodecyl sulfate-polyacrylamide gel electrophoresis for separating P-450~~~ from its derivatives and in the study of the smallest of which was readily accessible by sieve chro-the API-labeled heme peptides. Since the process of isoelectric matography but accounted for only 10 to 15% of the heme focusing imposes and maintains the resolution a supporting protein (30). Due to its strong immunochemical cross-reactiv-matrix of relatively low cross-linkage suffices which together ity with antibodies made against the parent protein (31) this with the absence of sodium dodecyl sulfate favors the retenheme peptide had gained great significance for comparative tion of heme. At the very low concentrations needed for studies of P-450 heme proteins (12). Application of the same electrofocusing in gel the solubilities of the API-derivatives of procedure of BrCN digestion to the photocovalent P-450~~~. P-450~~~ and its heme peptide are adequate while free soluligand complex, however, led to complete degradation of the heme protein and afforded the smallest heme peptide exclusively at the expense of the longer heme peptides. The cause of this fortuitous increase in yield to 80% is not yet clear but it could be due in part to an increase in stability of the heme chelation after covalent binding of the label. Binding of the label may also engender a small conformational change giving better access for BrCN degradation to the 2 methionyl residues surrounding the structural domain which includes both tion electrofocusing in density gradients presents solubility problems with the protein derivatives.
The second discrete binding site for API on cytochrome P-450cAM has significantly lower binding affinity so that a homogenous, monolabeled P-450~~~. ligand complex can be readily prepared with API. The binding affinity for API at this second site is, in turn, several orders of magnitude higher ' K. M. Dus, unpublished results.