THE ISOLATION AND PROPERTIES OF MICROSOMAL CYTOCHROME”

BY PHILIPP STRITTMATTER AND SIDNEY F. VELICK

(From the Department of Biological Chemistry, Washington University School

of Medicine, St. Louis, Missouri)

(Received for publication, October 12, 1955)

Mammalian liver contains a heme protein which from its spectrum and properties in particulate preparations has been termed cytochrome b, by Yoshikawa (I), cytochrome m by C. F. Strittmatter and Ball, who localized it in microsomes (2,3), and cytochrome bg by Chance and Williams (4). In view of the fact that this and other microsomal enzymes have been studied chiefly in particulate suspensions, methods of isolating microsomal pro- teins for more detailed characterization were examined. The first and most easily recognizable of the enzymes that we have obtained is the microsomal cytochrome. This paper deals with the liberation of the cytochrome from its particulate complex, the fractionation steps in its purification, electro- phoretic and ultracentrifugal tests for homogeneity, molecular weight de- termination, spectral properties, and reactions with a number of reagents.

Materials and Methods

Optical measurements were made in a Beckman DU spectrophotometer calibrated against emission bands of a mercury arc. Silica cuvettes of 1 ml. capacity and 1 cm. light path were used in the aerobic experiments, while, in experiments involving removal of oxygen or equilibration with another gas, cuvettes of the Thunberg type or the injection systems de- scribed in the following paper (5) were employed. Anaerobic conditions, in which no appreciable reoxidation of the cytochrome or reducing agents occurred in several hours, were obtained in such systems by alternately evacuating and flushing ten to twenty times with Linde nitrogen, and by using only solutions deoxygenated by bubbling Linde nitrogen through them for 30 minutes or longer.

Reduced di- and triphosphopyridine nucleotides (DPNH and TPNH, respectively), horse and beef heart preparations of cytochrome c, and re- crystallized tris(hydroxymethyl)aminomethane (Tris buffers) were prod- u&s of the Sigma Chemical Company. The Versene brand of ethylene- diaminetetraacetic acid was employed. The indigo dyes were prepared by Dr. P. W. Preisler by the method of Sullivan, Cohen, and Clark (6).

* This work was aided by a grant from the American Cancer Society on recommen- dation of the Committee on Growth of the National Research Council.

253

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254 ISOLATION OF MICROSOMAL CYTOCHROME

EXPERIMENTAL

Preliminary Experiments

Microsomal fractions isolated from rat, guinea pig, rabbit, and beef livers exhibited absorpt,ion spectra in deoxycholate-clarified suspensions that were very similar to those that have been described for rat liver preparations (3). Rabbit liver was selected as a favorable compromise with respect to size and the quantity of material that could be obtained. The method of Schneider (7) for isolating the microsomes, homogenization in 0.25 M

sucrose and differential centrifugation, was modified by centrifugally re- moving cellular debris, nuclei, and mitochondria in one step. This en- tailed a loss in microsomes which was more than compensated for by the gain in speed of processing large amounts of material. The factors which limited the scale of operations were the number and capacity of the high speed centrifuges. It was necessary to work batchwise with the livers of two rabbits at a time. The duration and force of the centrifugation em- ployed in separating the microsomes were greatly diminished by addition of ammonium sulfate to 0.4 saturation. This aggregated the microsomes and at the same time precipitated very little of the soluble liver protein. One washing with dilute buffer in the preparative ultracentrifuge was em- ployed to remove the major portion of this contaminant.

In a search of methods for isolating the microsomal proteins, acetone powders, butanol extraction, and treatment with ribonuclease, trypsin, and pancreatic lipase, both individually and in combination, were tried. Of these, only butanol extraction by methods similar to those of Morton (8) and lipase treatment yielded soluble preparations which contained the cyto- chrome, and by far t,he most consistent and successful preparations were obtained with pancreatic lipase. Incubation with lipase is used in Step 4 of the preparation. The pancreatic lipase, preliminary samples of which were supplied by Dr. R. K. Crane, was partially purified by fractional pre- cipitation and adsorption steps (9). Since addition of trypsin inhibitor to several lipase incubations did not inhibit liberation and since trypsin it- self did not liberate the cytochrome, this type of proteolysis, at least, is not involved. Also, the microsome suspensions undergo no appreciable autolysis on incubation at 37” for 1 hour, as measured by increase in nin- hydrin-positive material. The lipase preparations, however, do contain a detectable amount of undefined proteolytic activity.

Preparation of Microsomal Cytochrome from Rabbit Liver

Materials-A sucrose medium, containing 0.25 M sucrose and 0.001 M

Versene, pH 7.5, in glass-distilled water, is prepared in advance and pre- chilled in the cold room. All solid ammonium sulfate used in the prepara-

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P. STRITTMA’M’ER AND S. F. VELICK 255

tion is purified as follows: A saturated solution is prepared with reagent grade material. Ammonia is added to approximately pH 8 and the solu- tion is filtered. The filtrate, after addition of Versene to a concentration of 0.001 M, is concentrated by boiling and cooled to obtain crystals which are washed with cold glass-distilled water and dried at 100”. Saturated solutions of ammonium sulfate used in the preparations contained 720 gm. of reagent grade ammonium sulfate per liter of water and 3.8 ml. of con- centrated ammonium hydroxide per liter of solution. After filtration, Ver- sene is added to 0.001 M, the pH is adjusted to 7.5 with 1 N hydrochloric acid, and the solution is chilled. Thirty rabbits, weighing 5 to 7 pounds each, are sacrificed two at a time. All subsequent steps are carried out at 4-S’ unless otherwise specified.

Step l-Two rabbits are stunned and bled, and the livers excised and washed in cold sucrose media. After blotting and weighing, the livers (about 150 gm.) are minced well with scissors for 1 minute and homogenized for 20 seconds in a Waring blendor at high speed with approximately 3 vol- umes of sucrose media per weight of tissue. The homogenate is then di- luted with sucrose media to 7 volumes per weight of tissue and is centri- fuged immediately in Servall centrifuges at 80 to 82 volts (9000 X g) for 12 minutes. This procedure is repeated with successive pairs of animals. The total elapsed time for each pair of two rabbits is not more than 20 to 25 minutes. The volume of pooled supernatant fluid, containing the micro- somes and largely free of nuclei and mitochondria, is approximately 16 liters.

Step C-560 ml. of saturated ammonium sulfate solution are added to 800 ml. aliquots of the supernatant fluid from Step 1. After 5 minutes the suspension is centrifuged for 7 minutes in Servall centrifuges at 110 volts (11,000 X g). The pellets, containing the microsomes, are resus- pended in a small amount of Tris buffer, 0.1 M, pH 7.5. The total volume, on resuspending the pellets from the entire 16 liters of supernatant fluid from Step 1, should be approximately 1600 ml.

Step S-The 1600 ml. of concentrated microsome suspension are centri- fuged in the No. 30 rotor of a Spinco model L ultracentrifuge at 0” for 70 minutes at 30,000 r.p.m. (80,000 X g). The dark red pellets are resus- pended in cold 0.1 M Tris buffer, pH 7.5, to give a total volume of 1000 ml. The elapsed time up to this point is 11 to 12 hours, and the suspension may be allowed to stand overnight at 5”.

Step 4-100 ml. of the dialyzed lipase solution (9) are added to 1000 ml. of the suspension, and the mixture is gently stirred for 1 hour at 37” and then cooIed in an ice salt bath to 5”.

Step 5-Ammonium sulfate fractionation of the lipase-treated prepara- tion is carried out as indicated in Diagram 1,

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256 ISOLATION OF MICROSOMAL CYTOCHROME

Step 6-The microsomal cytochrome preparation from Step 5 is refrac- tionated at least two times by the procedure indicated in Diagram 2. The cytochrome obtained after the second reprecipitation, usually about 50 to

1050 ml. lipase-treated preparation

I Small ppt. (discarded)

160 ml. SAS; centrifuge

1150 ml. red-brown suspension

250 ml. SAS; centrifuge

Large ppt., partly floating

I- Large tan ppt. (discarded)

I Small ppt. (discarded)

I Large cream ppt. (discarded)

Filter through glass-wool plug

1150 ml. red opalescent supernatant

100 ml. SAS; centrifuge

1200 ml. red opalescent supernatant

170 gm. AS (pH kept at 7.5 with 1 N

NaOH during AS additions); centri- fuge

1200 ml. red opalescent supernatant

Filter through coarse filter paper

1200 ml. clear red suaernatant

I

170 gm. AS; centrifuge

Light pink ppt. (discarded) 1250 ml. clear red supernatant

,m 100 gm. AS; centrifuge

Pink ppt. (dissolved and re- 1300 ml. clear red supernatant

fractionated in same man-

ner) Add 1 N HCl dropwise with stirring to

pH 4.20 as measured by glass electrode

I Clear yellow supernatant (dis-

carded)

I Red ppt. containing 100 to 120 mg. mierosomal

cytochrome

(Dissolved in 30 to 50 ml. 0.1 M Tris, pH 7.5)

DIAGRAM 1. Step 5. Ammonium sulfate fractionation. All centrifugations are

at 11,000 X 9 for 10 minutes. Abbreviations: SAS, saturated ammonium sulfate; AS, solid ammonium sulfate.

70 mg., was used in the experiments described in this paper. It could be stored at 5” in 0.1 M Tris buffer, pH 7.5, for at least 2 weeks without change in spectra or enzymatic properties, or kept frozen at -20” for longer periods of time.

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P. STRITTMATTER AND S. F. VELICK 257

Properties of Microsomal Cytochrome

Absorption Spectra-Fig. 1 shows the absorption spectra of a typical preparation of microsomal cytochrome in the oxidized and reduced forms. The reduced spectrum in the visible region is the same with a wide variety of reducing agents (see “Oxidizing and reducing agents”). In the example illustrated, excess cysteine was selected as reducing agent because it causes no appreciable spectral interference at wave-lengths higher than 300 rnp. The millimolar absorption coefficients, E,,, were based upon heme content as described in the section on the calculation of E,, values. In the Soret region, a 10 rnp shift occurs on reduction similar to that obtained in suspen-

50 ml. cytochrome solution from Step 5 or once reprecipi- tated cytochrome

I I

250 ml. SAS, pH 7.5; centrifuge

Ppt . discarded Clear red supernatant

pH to 5.2 by dropwise addition of 1 N HCl; centrifuge

I Ppt . discarded Clear red supernatant

pH to 4.4 by dropwise addition of 1 N HCl; centrifuge

I I Light pink supernatant fluid con- Large red ppt. of microsomal cytochrome

taining some cytochrome and (dissolved in 0.1 M Tris, pH 7.5, and stored impurities or refractionated)

DIAGRAM 2. Refractionation of microsomal cytochrome. The pH measure- ments were made with dipping electrodes. All centrifugations were at 11,000 X q

for 10 minutes. Abbreviations: SAS, saturated ammonium sulfate; AS, solid am- monium sulfate.

sions of microsomes (2). The E,, value at the Soret maximum increases from 117 in the oxidized form to 171 in the reduced form. From 300 to 600 rnp, the spectra correspond closely to those reported by Appleby and Mor- ton (10) for the flavin- and heme-containing lactic dehydrogenase of yeast, although, as will be shown, the microsomal cytochrome contains no flavin. The absorption peaks at 526 and 556 rnp are in agreement with the maxima reported for microsomal suspensions (3). In addition, extension of spec- tral observations to the 300 to 400 rnp region shows broad absorption bands for the oxidized and reduced forms at 355 to 370 rnp and from 320 to 340 mp, respectively. These bands, although broad, are comparable with the 526 and 556 rnp peaks in intensity.

Identity of Porphyrin-The heme group of the cytochrome is readily and quantitatively removed from the protein by an acid acetone method similar

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258 ISOLATION OF MICROSOMAL CYTOCHROME

to that of U. J. Lewis (11). The coincidence of the absorption spectrum of the heme with that of ferriprotoporphyrin chloride, Fig. 2, suggests the identity of the two compounds and prompted us to use the extinction co- efficients reported by U. J. Lewis to make a quantitative determination of the isolated heme spectrophotometrically.

Analysis for Iron and Heme and Calculation of E,, Values--Various cytochrome preparations mere dialyzed against 100 to 200 volumes of 0.001 M Tris buffer, 0.001 M cysteine, pH 7.0, or glass-distilled water for 36 hours with three changes of water or buffer. The heme protein bonds were bro-

I I I I I I I I I

2oo - ____ Oxidized Reduced

300 340 380 Wave

Ept4hbo (rn5p) 540 580 620

FIG. 1. Absorption spectra of oxidized and reduced microsomal cytochrome. Op-

tical density readings were taken at 5 rnp intervals over the entire wave-length range and at 1 rnp intervals at each absorption maximum or minimum. The protein spec-

tra were read in 0.10 M Tris, pII 7.35; for reduction 9 pmoles of cysteine per ml. were

added. The control tube contained the same buffer and the same amount of cys- teine. EmM values were based on heme analyses by the method of Lewis (11).

ken at or below pH 1 with hydrochloric acid at room temperature, and the protein was precipitated with approximately 20 volumes of redistilled ace- tone. After removal of the colorless protein by centrifugation, the super- natant fluid and acetone washings were diluted to volume with acetone for spectrophotometry. As a check on the heme analyses, analyses on the acid-acetone method were run on recrystallized horse oxyhemoglobin. These analyses agreed within 5 per cent with the calculated value based upon the oxyhemoglobin absorption bands. Alkaline pyridine and cya- nide hemochromogen derivatives, prepared in the usual way without prior separation of the protein, gave bands in the proper position, but were not wholly satisfactory. The apparent molar absorption coefficients as

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P. STRITTMATTER AND S. F. VELICK 259

well as details of the spectra are influenced by the protein as shown by vari- ations reported for different heme proteins carrying the same heme group (12).

Iron analyses, with reduced iron and also Mohr’s salt for preparing stand- ard solutions, were carried out on the dialyzed heme protein by the method of Drabkin (13). The same method, involving wet ashing and ortho- phenanthroline for color development, was employed on the quantitatively isolated heme after evaporation of the acetone. The results by both pro- cedures were the same. A commercial preparation of cytochrome c, 0.34

I I I I I I I I , I I , I

2.00 - .20 - Points -Cytochrome hemin _ Solid line-Ferri-protoporphyrin

chloride 1.50 _ .I5 -

> .+$ B-

5 LOO- .z 5 .I0

n Cl 7-E . 5

.,!Ly%,, :

~~~50- l

.u

0 . 505

.

300 380 460 420 500 580 660 Wave Lenyth (m EJ)

FIG. 2. Comparison of absorption spectra of acetone-HCl derivative of microso-

ma1 cytochrome and ferriprotoporphyrin. The ferriprotoporphyrin chloride spec- trum according to Lewis (11).

per cent iron, gave concordant values by wet ashing and by ignition. The spectra of all solutions subjected to analysis were measured.

The millimolar absorption coefficients based upon iron content and upon heme are presented in Table I. The agreement between the values of E,, based upon heme and upon iron analyses is adequate. Variations of f8 per cent in the values based on iron and f4 per cent in the values based on heme may be, in part, of analytical origin. However, the different preparations may not be completely uniform. It is difficult to exclude small and variable traces of non-heme iron, and it is possible that a small amount of denaturation occurs during the acid precipitation step.

Dry Weight and lMinima1 Molecular Weight-In Table II are the results of dry weight determinations and the minimal molecular weights calculated

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260 ISOLATION OF MICROSOMAL CYTOCHROME

upon the basis of the dry weights and the E,, values of the reduced cyto- chrome at 423 mp. The protein samples, after prolonged dialysis against glass-distilled water, were dried in vacua at room temperature and then at 110’ to constant weight. Although these calculations are subject to the cumulative errors in E,,, in drying and weighing small samples, and in possible contamination by salts and residual water, the results are in agree- ment with those obtained from sedimentation and diffusion analyses as de- scribed in the section on molecular weight.

Sedimentation CoefJicient-Two solutions of the cytochrome, 0.2 per cent protein in 0.1 M acetate, pH 6.95, and 0.4 per cent protein in 0.1

TABLE I

Absorption Coeficients for Microsowud Cytochrome Based on Iron and Heme Analysis

Analysis

Iron 8 113 f 10

Heme 4 117 3z 5

No. of preparations

analyzed Oxidized, 413 ml

Enm of absorption peaks

Reduced, Reduced, 423 m,. 526 Inp

Reduced, 5.56 mp

IIOIl Hail

:ER5~13.4*o.5~21a*1.~ 1.04

TABLE II

Minimal Molecular Weight Based on Iron and Heme Analyses and Dry Weight

Preparation No. Opty&le$ity Cytochrome concentration* Dry weight Minim;~i;;ecular

-

/.lmole per ml. mg. ger ml.

19 10.3 0.058 0.942 I 16,200 44 31.6 0.185 3.55 19,200

* Based on E,, = 171 at 423 mp for the reduced form.

ionic strength phosphate, pH 7.38, sedimented with a single boundary in prolonged sedimentation runs (5 t,o 6 hours) at 5” in the analytical ultra- centrifuge. The resulk in phosphate, corrected to an average rotor tem- perature of 10” in water, give a sedimentation coefficient s~o,~, of 1.0 X IO-l3 see -l . .

Difficsion CoefJicient-The diffusion coefficient was determined with a 0.3 per cent protein solution in 0.1 ionic strength phosphate, pH 7.38, in duplicate. Roundaries were formed in both limbs of a standard Tiselius electrophoresis cell (Klett) and, after compensation, were followed at inter- vals with Longsworth schlieren scanning photographs at 2”. Normalized curves at four time intervals fell within close limits on the Gaussian distri- bution curve. The diffusion coefficients, DlO,W, calculated by the maximal

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P. STRITTMATTER AND S. F. VELICK 261

ordinate and the maximal ordinate-area methods (14), by using sixteen time points, are 5.57 X lo-’ and 5.48 X lo-’ cm.2 sec. -l, respectively. For comparison with sedimentation coefficients these measurements, made near 2”, were extrapolated to the conditions prevailing in water at 10”.

Partial Specific Volume-This quantity (7) was determined by the density gradient method (15) and found to be 0.748.

Molecular Weight-By using the above quantities in the standard rela- tion for molecular weight (14), a value of 16,900 is obtained for the molecu- lar weight. From this value and the partial specific volume, one may cal- culate the radius of the sphere of equivalent volume, and correspondingly the diffusion coefficient, Do, for the equivalent sphere in water. The ratio between this value and the observed coefficient is 1.7, indicating a high de- gree of molecular asymmetry.

Electrophoretic Analysis-Experiments were carried out in the pH range 6.9 to 7.5 with 0.1 M Tris, 0.1 ionic strength phosphate, and 0.1 M acetate buffers. The single ascending boundary in these experiments remained sharp and symmetrical for 150 to 300 minutes. The descending boundary broadened more rapidly and, after 3 to 4 hours, showed indication of two very slowly separating heme protein components in an area ratio of roughly 3:2. In view of the spectral evidence presented here and in following pa- pers (5,16), we consider the inhomogeneity to represent minor charge modi- fications of a single heme protein species. Although no evidence of a more fundamental inhomogeneity has been detected, the problem awaits more detailed investigation.

Over the limited pH range studied, the protein carried a relatively high, net negative charge. The mobility at pH 7.5, in 0.1 M Tris chloride buffer, is -5.9 X lo+ cm.2 volt-’ sec.?. In view of the mobilities and of the fact that the pH of water-dialyzed solutions becomes stabilized between pH 5 and 6, it is likely that the isoelectric and isoionic regions are below pH 6. It should be noted in this respect that the solubility of the protein in concentrated ammonium sulfate solutions is greatly diminished at pH 5 and below, a property utilized in the purification.

Flavin AnaZysisFlavin analyses were carried out by the sensitive flu- orometric method of Burch, Bessey, and Lowry (17). The results, for which we are indebted to Dr. Helen B. Burch, reveal no more than 0.5 X 10-d molecule of flavin per molecule of cytochrome. It is concluded that flavin does not form an integral part of this protein. This is in contrast to the yeast lactic dehydrogenase (10) which exhibits a similar spectrum but contains flavin and heme in a 1: 1 ratio.

Stability-At neutral and alkaline pH the cytochrome is stable. The spectrum remains unchanged for at least several hours at 20-37”, pH 7.5. Preparations have been incubated for 1 hour at 25” at pH 8.1 to 9.5 with

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262 ISOLATION OF MICROSOM.4L CYTOCHROME

no appreciable change in the oxidized or reduced spectra. Neutral solu- tions can be stored for at least 2 weeks at 5” and much longer at -20”. The protein is less stable, however, in the acid pH region. When samples of the cytochrome were incubated at 25” for 10 minutes at pH 6.5, 4.3, and 3.5, the amounts of denaturation as measured by loss of cytochrome spec- trum were 0, 30, and 100 per cent, respectively. This denaturation is very much depressed at low temperatures (5”) and at high salt concentration (90 per cent ammonium sulfate saturation), and it is for this reason that the acid precipitation of cytochrome in the isolation procedure entails little loss of material.

Effects of Vatious Reagents--N-Ethyl maleimide, 10e3 M, incubated with the protein at pH 7.4 for 24 hours at 5”, and p-chloromercuribenzoate, 1O-3 M, had no effect on the oxidized or reduced spectra in the 400 to 600 mp range, indicating that no thiol groups susceptible to these reagents affect the heme. Hydroxylamine, 0.1 M, pH 7.4, and hydrogen peroxide, 0.1 M, pH 8.0, were similarly without effect. Hydrogen peroxide, however, oxidized the reduced form. No significant effect on the oxidized or reduced spectra were obtained with 0.1 M sodium cyanide, pH 8.0, or in solutions kept in an atomosphere of carbon monoxide at pH 8.0 for 3 days at 5” or for 1 hour at 25”. Except where otherwise indicated, these reagents were incubated with the cytochrome for 15 to 30 minutes at 25” under anaerobic conditions. In view of the slight spectral effects observed by Horecker and Kornberg (18) with cytochrome c and sodium cyanide, a more detailed examination of this reagent with the microsomal cytochrome should be undertaken. It is especially noteworthy that carbon monoxide does not alter the absorption spectrum of either the oxidized or reduced form even on prolonged equilibration.

Oxidizing and Reducing Agents-The microsomal cytochrome at pH 7 to 8 is reduced by reduced indigo di-, tri-, and tetrasulfonates, anthra- quinone-2,7-disulfonate, and benzyl viologen, and by sodium hydrosulfitc and cysteine. Potassium borohydride at pH 5.5 is also an effective reduc- ing agent. In all cases the spectra obt’ained were identical, in those regions where comparison was possible, with that shown in Fig. 1. Large excesses of reduced glutat,hione caused no appreciable reduction. Neither DPNH nor TPNH alone reduces the oxidized cytochrome. Rapid and complete reduction of the cytochrome by these coenzymes is cat,alyzed by reductases that have been obtained in soluble form from microsomes. The spectrum of the cytochrome reduced enzymatically is the same as that observed with the various other reducing agents. The liberation of the TPNH enzyme and the preparation and properties of the DPNH-specific reductase are described in a following report (16).

Reduced microsomal cytochrome is oxidized rapidly by potassium fer-

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P. STRITTMATTER AND S. F. VELICK 263

ricyanide, ferric chloride, cytochrome c, and various dyes of appropriate potential. It is oxidized somewhat more slowly by oxygen and by mer- curic chloride to give the oxidized spectrum shown in Fig. 1.

DISCUSSION

The liberation of the microsomal cytochrome from its particulate com- plex appears to be primarily lipolytic, although the possibility of some pro- teolysis has not been rigorously excluded. In so far as comparison with the particulate pigment is possible (24), no significant alteration of proper- ties has been detected with the exception of the apparent standard poten- tial. This potential determination and its interpretation are treated in the following paper (5). The isolated protein, by spectral criteria, appears homogeneous with respect to its heme properties. Since the material sedi- ments as a single compound, the apparent electrophoretic inhomogeneity may result from minor charge modification of the same substance, similar perhaps to the heterogeneity of crystalline insulin which may vary in its content of amide nitrogen (19). The agreement between molecular weight and the minimal molecular weight based on iron and heme analysis indicates the presence of one heme per molecule and no significant amount of non- heme iron, and provides some stoichiometric evidence for homogeneity. Particular attention was paid to establishing molar absorption coefficients, since these provided the experimental basis for work to be described and since cytochrome c is the only other mammalian cytochrome for which such properties had been directly determined.

The spectra of the cytochrome, in both its oxidized and reduced forms, resemble those of other heme proteins which, on the basis of magnetic sus- ceptibility measurements, have been considered to coordinate iron by bonds of predominantly covalent character. The inertness to a variety of re- agents that attack heme iron is consistent with this classification. Failure to react with carbon monoxide during prolonged exposure is of particular significance in this respect. The protein is quite stable in the alkaline pH region and shows no spectral changes as a function of pH up to pH 10. Spectral changes in the acid pH region are associated with the breaking of the heme protein bonds, as indicated by the complete resolution of heme and protein by the acid acetone method. This behavior is in contrast to that of cytochrome c, in which the heme is bound to the protein by thio ether linkages and undergoes fully reversible changes down to pH 1.

SUMMARY

1. The cytochrome of rabbit liver microsomes has been isolated by treatment with pancreatic lipase and purified by ammonium sulfate frac- tionation at controlled pH.

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264 ISOLATION OF MICROSOMAL CYTOCHROME

2. The purified protein sediments in the ultracentrifuge as a single pro- tein. From sedimentation velocity, diffusion, and partial specific volume measurements, the molecular weight is about 17,000.

3. Quantitative analyses for iron and heme indicate the presence of one heme per molecule and no non-heme iron.

4. The spectrum of the isolated ferriporphyrin chloride in acetone is identical with that of the ferriprotoporphyrin chloride obtained from hemo- globin. Unlike cytochrome c, the heme and protein of the microsomal cy- tochrome are readily separated by acid acetone treatment.

5. The spectrum of the purified microsomal cytochrome, intermediate between that of cytochrome c and particulate mitochondrial cytochrome b, gives no evidence of more than one heme protein component, a finding supported by the stoichiometry and the sedimentation analysis.

6. Prolonged electrophoresis reveals the possible presence of two heme protein components distinguishable only by a very small difference in net charge.

BIBLIOGRAPHY

1. Yoshikawa, H., J. Biochem., Japan, 38,l (1951). 2. Strittmatter, C. F., and Ball, E. G., Proc. Nut. Acad. SC., 38, 19 (1952).

3. Strittmatter, C. F., and Ball, E. G., J. Cell. and Comp. Physiol., 43, 57 (1954). 4. Chance, B., and Williams, G. R., J. Biol. Chem., 209,945 (1954). 5. Velick, S. F., and Strittmatter, I’., J. Biol. Chem., 221, 265 (1956).

6. Sullivan, M. X., Cohen, B., and Clark, W. M., Bull. Hyg. Lab., U. S. P. H. S., No. 151, 57 (1928).

7. Schneider, W. C., J. BioZ. Chem., 176, 259 (1948).

8. Morton, R. K., Nature, 166,1092 (1950). 9. WillstB;tter, R., and Waldschmidt-Leitz, E., Die Methoden der Fermentfor-

schung, Leipzig, 1560 (1941). 10. Appleby, C. A., and Morton, R. K., Nature, 173,749 (1954). 11. Lewis, U. J., J. Biol. Chem., 206, 109 (1954).

12. Paul, K. G., Theorell, H., and Akeson, A., Acta them. Stand., 7, 1284 (1953). 13. Drabkin, D. L., J. Biol. Chem., I.&O, 387 (1941). 14. Neurath, H., Chem. Rev., 30, 357 (1942).

15. Linderstrom Lang, K., and Long, H., Compt..rend. trav. Lab. Curlsberg, &+ie chim., 21, 315 (1936).

16. Strittmatter, P., and Velick, S. F., J. Biol. Chem., 221, 277 (1956).

17. Burch, H. B., Bessey, 0. A., and Lowry, 0. H., J. BioZ. Chem., 175, 457 (1948). 18. Horecker, B. L., and Kornberg, A., J. BioZ. Chem., 166, 11 (1946). 19. Harfenist, E. J., and Craig, L. C., J. Am. Chem. Sot., 74, 3083 (1952).

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Philipp Strittmatter and Sidney F. VelickMICROSOMAL CYTOCHROME

THE ISOLATION AND PROPERTIES OF

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