Reversible inhibitions of cytochrome system components by macromolecular polyions

ARCHIVES

OF

BIOCHEMISTRY

Reversible

AND

BIOPHYSICS

Inhibitions

94,

(1961)

392-404

of Cytochrome

by Macromolecular PHILIP From

the Special

Dental

PERSON

AND

System

Components

Polyions ALBERT

Research Laboratory, Brooklyn, New

Veterans York

S. FINE Administration

Hospital,

Received February 6, 1961 Cytochrome oxidase activity of whole heart homogenates, Keilin and Hartree heart muscle particles, and deoxycholate-treated heart muscle oxidase preparations were all inhibited by the polycations, protamine (sulfate), histone, ribonuclease, and lysozyme. The polycationic inhibitions could be blocked and reversed by the polyanions, polyglucose sulfate, polyethylene sulfonate, carageenins, sulfochitosans, DNA, chondroitin sulfate, and heparin. Neither hyaluronic acid nor n-acetylneuraminic were effective in regard to blocking or reversing the polycation inhibitions of oxidase activity. Polyglucose sulfate and other macroanions, alone, were capable of increasing the cytochrome oxidase activity of non-protamine-treated enzyme preparations. Polyglucose sulfate also restored oxidase activity lost as a result of aging of enzyme preparations. Polyanionic substances including polyglucose sulfate, chondroitin sulfate, heparin and glutamyl polypeptides formed complexes with cytochrome c, with resultant loss in catalytic activity of the hemeprotein. Such complex formation could be reversed by addition of protamine to the system, with reactivation of cytochrome c. The reversible interactions between polyions and the cyt,ochrome system components were more effective in Tris than in phosphate buffers of equal molarities. Increase in ionic strength beyond 0.08, resulted in a weakening of the protamine inhibition of oxidase activity. It was concluded that coulombic attractions between cytochrome system component,s and oppositely charged polyions were involved in the phenomena described. INTRODUCTION

Observations

made during

histone inhibit. cytochrome oxidase activity of beef and rat heart muscle preparations. In addition, we could block and reverse the above-mentioned polycation inhibitions of cytochrome oxidase activity by means of polyanions including polyglucose sulfate, deoxyribonucleic acid (DNA), heparin, and polyethylene sulfonate. It was also found that polyglucose sulfate and other polyanions could, in low concentrations, enhance the activity of cytochrome oxidase preparations. Finally, it was noted that some of the above polyanions, when used in higher concentrations, caused decreases of cytochrome oxidase activity in the manometric and spectrophotometric assays employed. Investigation of this latter phenomenon has indicated that, at higher concentrations, the

comparative

studies of cartilage respiration indicated that substances present in cartilage homog-

enates might inhibit cytochrome oxidase activity of mammalian heart muscle preparations (1, 2). Based upon these observations, a survey of polyionic marcomolecules was begun, with the hope of finding previously unknown

inhibitors

and activators

of

cytochrome oxidase. While these studies were in progress, Smith and Conrad (3-5) reported that the basic proteins, salmine and cytochrome c, inhibited cytochrome oxidase activity of various preparations. We have confirmed the work of Smith and Conrad and have also shown that other macrocations such as ribonuclease, lysozyme, and 392

REVERSIBLE

CYTOCHROME

polyanions formed complexes with the polycationic cytochrome c of the assay systems, with resultant interferences in cytochrome oxidase activities. A preliminary report of this work has been published (6 j . MATERIALS Unless 0t11crwisc indicated, glass-redistilled water was used in all experiments. Rufer systems were KH2P0,-NaaHPOI (Sorensen’s phospham and HCl-2-amino-2-hydroxymethyl-1,3buffer) propane&o1 (Tris buffer). CYTOCHROME

0x1~~s~

PREPARATIONS

Fresh rat heart homogenates were prepared in water, using a ground-glass homogenizer (450-500 mg. wet weight tissue made t,o 100 ml. homogenate). Male albino rats of 200400 g. body weight, maintained on an ad libitum intake of Purina rat checkers and tap water, were used. Keilin and Hartree type beef heart muscle preparations were made (7, 8), with the modification that final suspension of the heart muscle particles was in 0.05 M Tris buffer, pH 7.4, instead of phosphate buffer. In experiments in which this type of preparation was used (see Figs. 8 and 9), the final concentration of the prrparation in the assay system was 0.091 mg. N/ml. I)eoxycholate-trecrted cytochTome oxidase preparations were made as described by Eichel et al. (9) and correspond to their “2-3” 1)rrparation. -4 single preparation, designated “2-3” XII, served as enzyme source in the experiment,s in which dcoxycholate-treated enzyme was used, except, as noted below for the ionic strength experiments. Tris buffer pH 7.4, 0.05 Ylf was used in suspending the Keilin and Hartree material from which this preparation was derived, and the same buffer was used during deoxycholate treatment. The final concentration of this preparation in the assay system reported was 2.18 X IO-” mg. N/ml. In o&r to st,udp the variables of pH and ionic strength, a second deoxycholate-treated preparation, “2-3” XIV was made, in which dcoxycholat~c trcatmrnt was carried out in water instead of buffer. The final concentration of this preparat,ion in the assay system was 1.76 X 1O-3 mg. N/ml. Both deoxycholate-treated preparations exhibited characteristic absorption spectra (9). Beef henrt cytochromc c (Sigma Chemical Co. Type V, NaCl and (NH,)SO, free) was used in thr cyt,ochromc oxidase assays. The cytochrome c was freshly made np in water for each experiment,. It was boiled for 5 min. prior t,o use in order to L’RL~ denatured cptochrome to precipitate, and then cooled and centrifuged to remove any pre-

893

INHIBITIOSS

cipitate that formed. Sodium dithionite WBF used as t,he reducing agent. Mncrocntions used in our experiments were : pro&mine sulfate’ (Nut,ritional Biochemicals Corp. and Sigma Chemical Co.) ; lysozymc~ (Sigma) : histone, calf thymus (Mann Research Labs., Inc.) and ribonuclease (Worthington Biochemical Corp.) Xncronnions were .sodium polyg1rrco.w sulfate, average molecular weight = 19,300, containing three sulfate groups per anhydroglucose unit, corresponding to preparation “H” (10) : chondroitin sUlfatc (Sigma, Worthington, Nutrit,ional, and General Biochemicals, Inc.) : chondrwitin sulfnte “C”; h-carageenin (Marine Colloids, Inc.) : IjSA (Sigma, calf thymus and Mann. calf thymu) ; rl-slLlfnchitoscrrL (Upiohn Co.) ; hcpnrirc, sodium U.S.P. (Fisher Scientific Co.) ; l~olyethylene sulfonate, average molecular weights = 12,900 and 5900 (IJpjohn Co.) ; hyuluronic ctcirl (Worthington); a series of v-glutamyl polypeptides of average molecular weight 2300, 10,600, 45,000 anct 162,000 (11). The above-listed polyions were ~~scti as obtained from their suppliers, without further treatment or characterization, other than pH adjustment of solutions when required. METHODS Cyt.ochrome oxidase activity was determined using st)andard spectrophotomrtric (12) and manometric (9) assays. Nitrogen was determined by means of a standard micro-Kjcldahl procedure (13). RESULTS

1. MA~R~CATION

INHIBITIONS OF CTTOCHROME 0x1~~~ ACTIVITI’, AND BLOCK AND REVERSAL OF IKHIBITIOXS By MACROANIONS

Inhibition. of Cytochronze Oxidase Activity by ProtamineL Cytochrome oxidase activities (spectrophotometric and manometric) of in) fresh rat heart homogenates, (b) Keilin and Hartree insoluble beef heart muscle preparations, and (c) deoxycholate-treated beef heart muscle oxidasc preparations were all inhibited by protamine. In a representative spectrophotometric ‘Since the active component of the protamine sulfate is the protaminc moiety, we hcncrforth refer to protaminc sulfate as protamine, with the understanding that in each instance it was rm~d RS the sulfate and not as the free 1x1s~.

394

PERSON AND FINE

!4.30 -Y/ml 2.86-t/m/

I.43

r/ml

0.377/m/ Control

1 I

I

I

2

3

MINUTES

FIG. 1. Inhibition of deoxycholate-treated beef heart muscle cytochrome oxidase activity by increasing protamine concentrations. Spectrophotometric assay curves shown are direct tracings of continuously recorded absorbancy changes. Assay: to a 1.0 cm. cuvette were added in the following order, 1.0 ml. of dithionite-reduced cytochrome c in water (= 1.0 mg. cytochrome c), 2.3 ml. of 0.05 M Tris buffer pH 7.4, 0.2 ml. of deoxycholate-treated cytochrome oxidase (Prepn. XII) in water. In the inhibitor addition experiments, 2.2 ml., instead of 2.3 ml. buffer was used, and 0.1 ml. inhibitor in buffer (to yield the final concentrations shown) was added immediately prior to enzyme addition Temp. 25”C., k = 550 rnk, slit width = 0.05 mm. (The wavering line of the curves reflects turbidity and refractive changes in the system which result from interaction between the oppositely charged polyions). (pg./ml. = r/ml. in figure.)

experiment summarized in Fig. 1, increasing concentrations of protamine were used to inhibit deoxycholate-treated beef heart cytochrome oxidase activity. The per cent inhibitions produced by the varying concentrations of protamine, on the basis of the 3min. decrease in absorbancy of reduced cytochrome c were 0.37 pg./ml. 10% ; 0.72 pg./ml. 26%; 1.43 pg./ml. 58%; 2.86 pg./ml. 92% ; 14.30 pg./ml. 100%. In Fig. 2 is shown the effect of increasing protamine concentrations upon oxygen utilization of a fresh rat heart homogenate. In this experiment 13, 26, and 52 pg./ml. protamine produced 13, 25, and 70% inhibitions, respectively, of oxygen utilization by the heart muscle homogenate. One hundred per cent inhibition of manometric activity (not shown in Fig. 2) was readily obtained by increasing the protamine concentration, as has been demonstrated elsewhere (6) and also later in this article. Nitrogen analyses performed to establish inhibitor nitrogen to enzyme nitrogen ratios, revealed a variable stoichiometry of

protamine inhibition with the different types of enzyme preparation. Thus, at 92% inhibition of the deoxycholate-treated enzyme activity (Fig. 1)) the protamine nitrogen to enzyme was 1.2:1. In the rat heart homogenate experiment (Fig. 2) at 73% inhibition, the ratio of protamine nitrogen to homogenate nitrogen was much higher, i.e., 2.3: 1. In general, less protamine was required to produce 100% inhibition of deoxycholate-treated preparations than either of the other enzyme sources. Influence of Ionic Strength, pH and Nature of the Ruffer upon Protamine Inhibition of Cytochrome Oxidase Activity In the experiments to be described in this section, conkibution to the ionic milieu by the enzyme preparation was minimized by employing a deoxycholate-treated cytochrome oxidase in whose preparation water rather than buffers was used (Prepn. “2-3” XIV). Protamine inhibition of oxidase activity was strongly influenced by ionic strength,

REVERSIBLE

CYTOCHROME

INHIBITIONS

200 -

Contra/ I 80 -

/3 Y/m/

I 60 -

60 52 Y /ml 40

0

IO

20

30 MINUTES

40

50

60

FIG. 2. Effwts of increasing protamine concentration upon inhibition of cytochrome osidase act,ivity, as measured by oxygen ut,ilization of fresh rat heart homogenate. Manometric assay: Body of flask: 1.1 ml. of 0.1 M PO, buffer, pH 6.96; 0.25 ml. of 0.1 :W semicarbazideHCl, pH 6.90; 0.05 ml. water; 1.0 ml. homogenate. Center well: 0.2 ml. of 10% NaOH plus filter paper. Side arm: 0.3 ml. hydroquinone (= 3 mg.); 0.1 ml. cytochrome c (= 1 mg. cytochrome c). Total volume = 3.0 ml. Gas phase: air temp.: 37°C. In inhibitor systems, protamine was added to body of flask in 0.1 ml. buffer, keeping constant total \~olumr bJ adjustment of initial buffer addition. (pg./ml. = r/ml. in figure.)

as shown in Fig. 3. The open circle curw in Fig. 3 shows the effects of ionic strength upon the activity (left ordinate) of control, non-protamine treated, deoxycholate enzyme preparation (activity expressed as 3min. decreased in absorbancy of reduced cytochrome c at 550 mp.). The closed circle curw of Fig. 3 represents per cent inhibition (right’ ordinate) of the control enzyme activities, produced by 7 pg./ml. final concentration of protamine, at the ionic

strengths shown. Between ionic strengths 0.03 and 0.08 there was an opt’imum plateau for protamine inhibition of oxidase activity. Beyond 0.08, increasing ionic strength result’ed in a progressively marked loss of protamine’s inhibitory act’ion. Observations made early in our work indicated that the inhibitory effect of protaminc was more pronounced in Tris buffers t,han in equimolar phosphate buffers. This led to expcrimcnts in which the inhibition

396

PERSON

AND

FINE

pH range studied was in a relatively area on the acid side.

narrow

Block and Reversal” of Protamine Inhibition of Cy tochrome Oxidase Activity by Polyglucose Sulfate 2 320

t

I,,

II 10

I

20 IONIC

%

30

I

40

I

II

50

STRENGTH

FIG. 3. Effect of ionic strength upon protamine inhibition of cytochrome oxidase activity. The lowest ionic strength represented, i.e., 0.033, is that of the phosphate buffer used, alone. Subsequent increases in ionic strength were produced by addition of NaCl solutions. Assay method same as given in legend for Fig. 1, except, that phosphate buffer was used.

produced by a given concentration of protamine was compared in equimolar Tris and phosphate buffers. In a typical spectrophotometric experiment, the inhibition produced by 18 pg./ml. protamine in a 0.05 M phosphate buffer medium was 73%, whereas in 0.05 M Tris buffer, the same amount of protamine produced 100% inhibition. This difference in buffer influence will be referred to again in connection with the block and reversal of protamine inhibition by polyglucose sulfate. While polyion interactions are generally very sensitive to pH, in the case of the protamine inhibitions there was only a minimal effect of pH variation upon its inhibitory potency. To cite an example, in a spectrophotometric experiment using 0.05 M PO4 buffer: at pH 5.7 18 pg./ml. protamine inhibited 85% of the oxidase activity of the system. As the pH was increased at intervals of 0.3 or 0.4 pH units, the percentage inhibition slowly declined in a straight line curve to 72% at pH 7.7. Thus, there was only a decline of 15% in the inhibition during an increase of 2 pH units. This minimal effect is understandable in view of the high pR value of protamine and the fact that the

Protamine, 28 pg./ml., was used to produce 100% inhibition of the deoxycholatetreated oxidase preparation. A concentration series of polyglucose sulfate was then employed to block this inhibition, as shown in Fig. 4. The lowermost curve shows the activity of control, non-protamine-treated enzyme. The horizontal curve third from the top, shows 100% inhibition of control activity by 28 pg./ml. protamine. The remaining curves, from top to bottom of Fig. 4, show the effects of successively incorporating into the system, 7, 10, 15, 21, and 28 pg./ml. polyglucose sulfate to block the protamine inhibitions. It may be seen that 21 pg./ml. polyglucose sulfate completely blocked the protamine inhibition. Similar blocks of protamine inhibitions by polyglucose sulfate were demonstrated using Keilin and Hartree beef heart muscle preparations and fresh rat heart homogenates as enzyme sources. Experimental data demonstrating reversal of protamine inhibition by polyglucose sulfate were also obtained and have already been published (6). Influence of Ionic Strength and Buffer System on Polyglucose Sulfate Block and Reversal of Protamine Inhibition of Cytochrome Oxidase Activity Earlier in this paper it was shown that ionic strength and the nature of the buffer ‘Although the mechanisms involved are considered to be the same, a distinction is made bctween block and reversal of inhibition. When protamine interacted with polyglucose sulfate prior to addition of enzyme, the polycation inhibition was blocked. If, however, protamine and enzyme interacted first, followed by addition of polyglucose sulfate after several minutes, the protamine inhibition was reversed. In the case of polyglucose sulfate, block and reversal of polycation inhibition were equally efficient. In the case of other polyanions such as DNA and chondroitin sulfate, block of polycation inhibition was more readily accomplished than was its reversal,

REVERSIBLE

CYTOCHROME

397

INHIBITIOSS 28 VPwt.

28 v/m/

Prof.

28VProt.

+15V

28 V Pro). 28 V Plot. \

t 77 PG6

PG.5

l PIVPGS t 28VPGS

Control

MINUTES

FIG. 4. Block of protamine inhibition of cytochrome oxidase activity by concentration series of polyglucose sulfate. Curves shown are direct tracings of continuously recorded absorbancy changes. For method, see legend, Fig. 1, modified as follows: Include immediately prior to addition of enzyme, addition of 0.1 ml. polyglucose sulfate in buffer (to yield pg./ml. concent,rations shown). (pg./ml. = r/ml. in figure.)

exert a strong influence upon protamine inhibition of cytochrome oxidase activity, and reference was made to similar effects upon polyglucose sulfate block and reversal of protamine inhibition. Thus, using deoxycholate-t.reated enzyme preparation in a 0.1 M phosphate buffer, 28 pg./ml. protamine produced 74% inhibit’ion, which was reduced to 50% inhibition by incorporation of 28 pg./ml. polyglucose sulfate in the system. However, in 0.05 M phosphate buffer, the same 28 pg./ml. protamine produced 100% inhibition of t’he same oxidase preparation, and addition of 28 pg./ml. polyglucose reduced the inhibition to only 20%. Similarly, polyglucose sulfate reversal of protamine was more effective in Tris than in equimolar phosphate buffer. Since there is a narrow pH range in which Tris and phosphate buffers overlap effectively, it was not possiblc to study the differential buffer effect over a broad pH range. The effect, was verified repeatedly, however, using equimolar Tris and phosphate buffers at pH values close to 7.4 and 7.7. Other lliacrocation. Inhibitions of Cytochrome Okdase Activity and Their Reversal 0 y Macroanions In addition to protamine, it was found that lysozymc, calf t’hymus histone, and

ribonuclease inhibited cytochrome oxidase activity. In Table It representative data are summarized for the four inhibiting macrocations named above. In terms of absolute amounts, protamine was the most effective inhibitor while ribonuclease was least effective. Lysozyme and histone were apparently equal in inhibitory potency, which TABLE MACROCATION

OXIDASE

(For assay method Substance

I

INHIBITTION~

OF

see legend for Fig. 1) Concn. ~~.,il.

Protnmine

CYTWAROME

ACTIVITY

0.37 0.72 1.13 2.86

Inhibition w,I

10 26 58 na

Lysozyme

‘28 56

18 71

Histone

33 66

35 80

33 100 200 333

17 35 50 78

Ribonuclease

398

PERSON

was less than that of protamine but greater than that of ribonuclease. It must be stressed that this order of activities is provisional, since all of the substances were used as obtained from the commercial suppliers, without additional treatment, standardization, or physicochemical characterization in regard to molecular weight and charge properties. We have therefore refrained from expressing relative inhibitory potencies on a molecular weight basis and report only the absolute concentrations employed. Macroanions other than polyglucose sulfate proved capable of blocking and reversing the protamine inhibition of oxidase activity. As shown in Table II, polyethylene sulfonate, h-carageenin, sulfochitosan, DNA, and chondroitin sulfate were effective in so doing. The first four substances listed above, i.e., polyethylene sulfonate, DNA, h-carageenin, and sulfochitosan, proved capable of effectively blocking prot’amine (33 pg./ml.) inhibition when used in concentrations of 33 or 66 pg./ ml. In the case of chondroitin sulfate, however, much larger amounts, i.e., 333 pg./ml. were not nearly as effective. Heparin behaved similarly to chondroitin sulfate in this respect. Hyaluronic acid and n-acetylTABLE

II

Enzyme activity determined by spectrophotometric assay according to method given in legend for Fig. 4, except that 0.1 M POa buffer was used. Concentration of protamine as inhibitor was 33 pg./ml. in each experiment. Macroanion concentrations varied as shown in col. (b) of the table.

-

-

(4

Per cent inhibition of oxidase by

Pe!L

(b)

inhibition

COW central

33 fig/ml.

of oxidase Iby 33pg./ml protamine

tion

protamine + macroanion EOIICII. shown in (b)

ug./ml.

Polyethylene sulfonate DNA X-Carageenin N-Sulfochitosan Chondroitin sulfat,e “C”

66

83

66 33 33 66 333

78 83 86 86 86 -

FINE

neuraminic acid were incapable of either blocking or reversing macrocation inhibitions when used at concentrations as high as 333 pg./ml. in the same test system. In regard to relative efficiency of block or reversal of protamine inhibition by the compounds listed in Table II, our experiments indicated that like polyglucose sulfate, compounds the polyethylene sulfonate, h-carageenin, and N-sulfochitosan were equally effective when either blocking or reversing protamine inhibition. However, the DNA preparations, the chondroitin sulfate preparations, and the heparin preparations employed were not nearly as effective in reversing protamine inhibition as they were in blocking it. Thus when a given concentration of DNA, chondroitin sulfate, or heparin interacted with a given concentration of protamine prior to addition of enzyme, the restoration of the activity of the system was greater than that obtained when the prot’amine first reacted with the enzyme, followed by addition of the macroanion. Protamine nucleinate did not influence activity in any way when added to t’he various oxidase preparations used. 2.

POLYGLUCOSE CPTOCHROME TREATED,

MACROANION BLOCK OF PR~TAMINE INHIBITION OF CYTOCHROME OXIDASE ACTIVITY

(a) Macroanion

AND

i

68 56

SULFATE

ENHAKCEMEST

OXIDASE ACTIVITY IX HEAT-TREATED

OF

IN UN-

AND

IN

AGED ENZYME PREPARATIONS

It, was often observed that if an effective excess of polyglucose sulfate over protamine was added to a system to block or reverse protamine inhibition, resulting enzyme activity might be greater than that of nonprotamine-treated controls. In other words, better than 100% reactivation was accomplished. The possibility therefore existed t’hat polyglucose sulfate, acting alone, might be capable of enhancing cytochrome oxidase activity. In Fig. 5 are shown results of an experiment in which 28 pg./ml. polyglucose sulfate produced a 40% increase in the activity of a deoxycholate-treated oxidase preparation. In extension of the above type of observation, we found that polyglucose sulfate added to deoxycholate-treated cytochrome oxidase preparations which had been subjected to heat denaturation, enhanced activity of the heat-treated material. In Fig. 6,

REVERSIBLE

CYTOCHROME

399

INHIBITIONS

\

200

Enzyme

+

56

PGS

7 /ml

FIG. 5. Enhancement of cytochrome oxidase activity by polyglucose sulfate addition. Curves shown are direct tracings of continuously recorded absorbancy changes. Assay method same as given in legend for Fig. 1, except that polyglucose sulfat,e was added instead of protamine SUlfiLte. (pg./ml. = r/ml. in figure.)

440

400

Key.’

q -PGS

1 360

t

=x &

+ PGS

1 320’.

120t SO 40 I

0 ‘-

II dda IO

TIME

FIG. 6. Effect oxidase. Enzyme varied between in an ice-water method same as tion for protamine

IN

15 MINUTES

20

25

30

of golyglucosc sulfat,e addition upon activity of heat-treated cytochrome was in solution in 2X glass-distilled water. Temperature during heating 44.5 and 45.O”C. Alifluots removed at 5-mm. intervals were rapidly chilled slurry. LUl determinations of enzyme activity wpre made at 25°C. Aasa> that given in legend for Fig. 1, with substitution of ~~ol~gluc~orc~ sldfatc addiaddition.

400

PERSOY

data from a typical experiment are shown. It may be seen that both for nonheated (0 time) and for heated enzyme, addition of 33 pg./ml. polyglucose sulfate consistently caused increases in oxidase activity which were quite reproducible. However, if polyglucose sulfate was added to an enzyme preparation prior to, instead of following heat treatment, results were erratic, and the activity-enhancing action of polyglucose sulfate on the heat-treated enzyme, as seen in Fig. 6, could not be demonstrated reproducibly. Addition of polyglucose sulfate to boiled, totally inactive enzyme was without any activity restoring effect. Several experiments were performed to ascertain the possible effect of polyglucose sulfate in reactivating aged cytochrome oxi-

AND

FINE

dase preparations. In a typical experiment, deoxycholate-treated oxidase was allowed to stand in 2~ distilled water for 27 hr. at 2-4”C., followed by 28 hr. at 25”C., following which its activity, as determined by spectrophotometric assay, was 25% of that exhibited by the freshly prepared enzyme. Addition of 100 pg./ml. polyglucose sulfate restored the activity to 100% of the original. Of three such experiments performed, in one experiment better than 100% restoration of activity was achieved. If, however, polyglucose sulfate was added to the oxidase preparation, prior to aging, so that the enzyme aged in the presence of the polyanion, then the loss of activity was greater than that which occurred when enzyme was allowed to stand without polyglucose sulfate. In such instances, further additions of polyglucose sulfate did not enhance or restore enzyme activity. In preliminary experiments, it was also found that like polyglucose sulfate, other macroanions shown in Table II were capable of enhancing the activity of cytochrome oxidase preparations. Thus ,V-sulfochitosan and chondroitin sulfate “C,” in concentrations of 33 and 66 pg./ml., increased the activity of deoxycholate-treated oxidase preparations by 2030%. 3. MACROANION INTERFERENCESIN CYTOCHROMEOXIDASE ASSAY

.I00

0.5

1.0

Ii-1 1.5

2.0

2.5

3.0

MINUTES

FIG. 7. Effect of concentration series of polyglucose sulfate on cytochrome oxidase activity. Sssay method same as given in legend for Fig. 1, except that polyglucose sulfate was added instead of protamine sulfate, and the reaction was followed in the Beckman DU spectrophotometer. The ordinate value of each plotted point is the observed absorbancy minus the absorbancy of totally oxidized cytochrome c. At 33, 66, and 100 pg./ml. concentrations, polyglucose sulfate activated the system. At 133 pg./ml. control activity was obtained. Above 133 pg./ml., inhibition of activity was produced. (pg./ml. = r/ml. in figure.)

It is both interesting and significant that macroanions which are capable of blocking and reversing the macrocation inhibitions of cytochrome oxidase, and also of enhancing the activity of cytochrome oxidase, may at higher concentrations lessen the activity of cytochrome oxidase assay systems. This may be seen in Fig. 7, in which is shown the effect of a concentration series of polyglucose sulfate upon the activity of a deoxycholate enzyme preparation. The third curve from the top represent’s the activity of the control, untreated, deoxycholate preparation. It may be seen that incorporating 33 and 66 pg./ml. polyglucose sulfate into the system produced increases in enzyme activity of 47 and 59%, respectively. As the concentration of polyglucose sulfate increased to 100 pg./ml., activation of the

REVERSIBLE

CYTOCHROME

system decreased to 29%; and with a concentration of 133 pg./ml., control activity was obtained, i.e., only 5% activation. Further increases in polyglucose sulfate concentration to 200 and 266 pg./ml. now produced inhibitions of 37 and 53%, respectively, of the activity of the system. These data, and also manometric experiments show that’ at lower polyglucose sulfate concentrations, oxidase activity was enhanced, as previously seen in Fig. 5. At the higher concentrations of polyglucosc sulfate, oxidase act,ivity was inhibited. The high concentration polyglucosc sulfate inhibitions could be blocked and reversed by protamine, as shown in Fig. 8. As may be seen in Fig. 8, the oxygen consumption of a control deoxycholate-treated oxidase preparation was -83 &/hr. Incorporation of 1 mg./ml. polyglucose sulfate to the system produced 50% inhibition. When 1 mg./ml. protamine was added to the polyglucose sulfate-containing system, the extent of inhibition was lowered to only lo”/;. The bottom curve shows that 1 me;./ ml. protaminc, when added alone to the enzyme system (in the absence of polyglucose sulfate) produced complete inhibition of oxygen consumpt~ion. In addition to polyglucose sulfate, chondroitin sulfate and heparin in mg./ml. concentrations also caused decreases in over-all oxidase activity in spectrophotometric and manometric assays, using all three types of enzyme preparation. It was possible to demonstrate the role of increasing molecular weight of polyanions upon their interference with activity of the oxidase system through the use of a series of y-glutamyl polypeptides of increasing average molecular weights. Results of these experiments are summarized in Fig. 9, in which per cent inhibition of oxygen utilization by a Keilin and Hartree beef heart muscle preparation is plotted as a function of molar concentration of the different molecular weight polypeptides used in the study. It may be seen that lesser concentrations of the heavier peptide units produced significantly greater inhibitions of oxygen utilization than did higher concentrations of the lighter peptides. In regard to ot,her anionic substances of

INHIBITIONS

401

100 I

MINUTES

Fro. 8. Protamine block of polyglucose sulfate interaction with cytochrome c. Details of manometric assay system similar to those given in legend for Fig. 2, except that source of enzyme was deoxycholate-treated beef heart preparation instead of rat heart homogenate. Details in text. IOOr z

90

t= -

*’

9

70

z_

6.

.\”

b M.W. M.W /62,000 45,000 -\

‘.

M.W.16

’

000

t

X IO-‘M

9. y-Glutamyl polypeptide inhibition of oxygen utilization. Influence of increasing molecular weight of polypeptide. Manometric assay as given in legend for Fig. 2 with modifications t,hat~ (2 ) deoxycholat,e-treated beef heart cytochrome oxidase and not rat heart homogenate was the enzyme source and (2) y-glutamyl polypeptide was inhibiting agent (instead of protamine). FIG.

biological interest, it should be noted t,hat neither hyaluronic acid nor ,%‘-acetylneuraminic acid possessed this type of inhibitory action, when used in concentrations of up to 5 mg./ml. We interpret the interferences in oxidasc activity by the relatively high polyanion concentrations to result from complex formation between the polyanions and the polycationic cyt’ochrome c. At the concentrations used in our experiments, such complexes form turbidities visible to the

PERSON AND FINE

402

unaided eye. Pertinent to these considerations are the experiments of Mora and Young (14), who reported the interaction of polyglucose sulfate and cytochrome c to form a precipitate which could be dissolved at pH 7 and which showed electrophoretic comigration of the macroanion and macrocation as a single complex, without dissociation. DISCUSSIO?;

The reversible inhibition of cytochrome oxidase activities by means of oppositely charged polyelectrolytes and the progressive weakening of the inhibitions in media of increasing ionic strength indicate that coulombic attractions between the polyions and the components of the terminal respiratory complex must be involved. The greater effectiveness of the polyion interactions in Tris than in phosphate buffer supports this contention. The bivalent-ion species of the phosphate buffer and its more complete dissociation undoubtedly exert a stronger effect on the polyions and “screening” thereby weaken coulombic interactions between them (15). The minimal effect of pH variation upon protamine inhibition of oxidase activity is understandable in view of the high pK value of protamine and in view of the fact that the pH values employed in our inhibition experiments were in a relatively narrow range several pH units below t’his. The above observations are of interest in relation to the recent work of Marks and McIlwain, who found that cerebral slices kept for 5-17 hr. at 0°C. lost their normal increase in respiration in response to electrical pulses (21). This loss of respiratory response was traced to liberation of histone from the cerebral cell nuclei and its interaction with the cytoplasmic respiratory apparatus of the cells (23). Significantly, the loss of respiratory response was greater in Tris than in phosphate buffer, and could be reversed by adding to the system the blood globulin fraction IV-4 of Cohn et al. (22). Our experiments suggest that the loss in respiratory response (i.e., oxygen consumption) by cerebral slices, and the blood globulin reversal thereof reported by McIlwain and co-workers, may result from

histone-cytochrome oxidase system interactions of the type described in this paper. Both cytochrome c solutions, and the deoxidase preparations oxycholate-treated formed visible complexes with polyanions and polycations, respectively. In the case of cytochrome c, complex formation was apparently associated with loss of its catalytic activity, as demonstrated in the manometric oxidase assay (Fig. 9). Recent unpublished studies in this laboratory have shown that polyglucose sulfate complex formation produces a marked increase in autoxidation rate of cytochrome c, a significant finding, since increased autoxidizability of cytochrome c is associated with loss of its catalytic activity (16). This polyanion-induced increase in autoxidizability may be related to the low molecular weight anion-induced autoxidizability of cyt’ochrome c, reported by Boeri and Tosi (17)) who showed that at acid pH values, the anions chloride, sulfate, fumarate, succinate, chloroacetate, trichloroacetate, and especially monoiodoacetate produced reversible increases in cytochrome c autoxidation. The question of whether the polycationic inhibitions of cytochrome oxidase activity result from interactions with t’he oxidase, per se, or with other components of the respiratory complex must remain unanswered at this time. In repeated attempts, it has not been possible to demonstrate alterations in the specific absorption spectrum of the oxidase during interactions with inhibiting polycations. Smith and Conrad (5) suggested that oxidase activity inhibition by polycations results from combination of the polycations with non-oxidase proteins contained in the terminal respiratory aggregates and does not involve interaction with the oxidase per se. They believe that although the oxidase itself does not react with the polycations, it is nonetheless masked by the complexed polycations, and thereby prevented from reacting with cytochrome c in solution. Smith and Conrad also referred to the work of Gamble (18) who showed that liver mitochondria and mitochrondrial fragments, in media of low ionic strength, are capable of binding with cytochrome c and salmine to form visible aggregates. However, Smith and Conrad dis-

REVERSIBLE

CYTOCHROME

counted such binding as being involved in their own work, since visible binding was apparently not observed by Gamble in phosphate buffers comparable to t,hose used by the former workers. The formation of visible aggregates cannot be used as the sole criterion of complex formation, since, as was mcnt,ioned earlier, Mora and Young ( 14) have shown that the insoluble complex formed by cytochrome c with polyglucose sulfate may be readily solubilized at neutral pH in phosphate buffer, and yet the complex behaved as a single unit which migrated without dissociating in an elcctrophoretic field. The possibility that cytochrome oxidase itself reacts with the polycations cannot be ruled out at present. We interpret polycation inhibit,ions of oxidase activity to mean that anionic sites on the oxidase, or on other components of the terminal respiratory system, or both, must be the attraction sites for interaction with the polycations, and that in some unknown way as a result of such int,eraction, electron transport is inhibited. The activation of cytochrome oxidase preparations by polyanions is also of significancc and suggests that polycationic substances of an inhibitory nature may be carried along with, and be present in, many of the oxidasc preparations currently used. The necessity for consideration of this type of inhibitory influence in regard to dcterminations of cytochrome oxidase activity has already been atrcssed by Smit’h and Conrad (5). One must also consider the possibility that the polyanion increases oxidaxe activity by virtue of other, as yet of unknown mechanisms. The reactivation cryed oxidasc preparations by polyglucose sulfate also deserves mention and suggests that loss of a preparation’s oxidase activity which occurs on standing may result in part from increased interaction with cationic groups in the preparation. The partial restoration in activity of heated oxidase preparations by polyglucosc sulfate is also interprctcd to result from combination of the polyanion with endogenous cationic inhibitors, and not necessarily from a reversal of heat tlenaturation of the enzyme. The latter possibility also exists, howvcvcr, and additional experiments arc‘ rquircd. The above

INHIBITIONS

403

observations have an important bearing upon the phenomenon first described by Keilin and Hartree, who found that the loss in succinoxidase activity of heart muscle particulates could be restored by the addition of crude albumin and also calcium phosphate gels to the assay system (19). Similar observations in regard to cytochrome oxidase activity were made by Horei (20) who used denatured globulin to restore oxidase activity. These phenomena were explained on the basis of “mutual accessibilities” and “spatial orientations” of catalysts and substrates (19). It is now obvious, we believe, that Ynutual accessibilit’ies” and “orient,ing influences” may be redefined in part, in terms of coulombic attractions betw-ctln polycations and polyanions. ACKNOWl,EDCME_VTS We are greatly indebted to the following individuals for their gcneroaity in providing samples of compounds used in this study: Dr. Max Ho\-arnick and Mr. Daniel T. O’Connell. of this y-glutamy polyept ides ; Dr. hospital, Karl Meyer, College of Physicians and Surgeons, Columbia IJniversity, chondroitin sulfates; Dr. Petri T. Mora, National Cancer Institute, polyglucosr sulfak ; Dr. Saul Rosrman, University of Michigan School of Medicine, ,\:-ac~t~t,~lnrur;~minic acid; Mr. I,eonard Stoloff, Marine Colloids, Inc., carageenins; and Dr. Donald T. Karnrr, The Upjohn Co., I)olyethylene sulfonates and ,qulfochitosans. \\-e wish also t,o express our thanks to Dr. PeteI, T. Morn for helpful and enlightc~ning tliscussion~. REFrmENCES 1. kkRSOS,

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FINE K-G., in “Methods in Enzymology”, (S. P. Colowick and N. 0. Kaplan, eds.), Vol. II, p. 749. Academic Press, New York, 1955. BOERI, IX., AND Tosr, L., Arch. Biochem. Biophys. 52,83 (1954). GAMBLE, J. I,., Riochim. et Biophys Acta 23, 306 ( 1957). KEILIX, D., AND HARTREE, E. F., Biochem. J. 44, 205 ( 1949). BOREI, H., Biochem. J. 47,227 (1950). MARKS, ?J., END MCILX-AIN, H., Biochem. J. 73, 401 (1959). COHN, E. J., STRONG, L. E., HUGHES, W. L., MULFORD, D. J., ASHWORTH, J. N., MELIN, M., AND TAYLOR, H. L., J. Am. Chem. Sot. 68,459 (1946). MCILWAIN, H., Biochem. J. 73, 514 (1960).

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