Sulphydryl blockage, haem oxidation, and the state of aggregation of Bustcon canaliculatum (gastropoda, mollusca) myoglobin

1st.3. Biochem.,1971,

2,

293-306.

[Scientechnica

(Publishes)

Ltd.]

SULPHYDRYL BLOCKAGE, HAEM OXIDATION, OF AGGREGATION

AND THE STATE

OF BUSYCON CANALICULATUM

(GASTROPODA, MOLLUSCA) JAN

293

P. JOHNSON

AND KENNETH

MYOGLOBIN R. H. READ

Biological Science Center, Boston University, Boston, Massachusetts,.oznr5; New England Aquarium, Boston, Massachugetts 021 IO; Department of Mollusks, Museum of Comparative Zoology, Cambridge, Massachusetts 02 I 38, U.S.A. (Received 7 Aug., rg7o) ABSTRACT I. Reaction of native dimeric Buzyconcanaliculatum myoglobin with fihydroxymercuribenzoate (PMB), JV-ethymaleimide (NEM), s,s’dithio(bis-2-nitrobenzoic acid) (DTNB), or iodoacetamide cleaves the native dimeric molecule into its 2 identical subunits, and converts the haem iron to the ferric state. 2. Reaction of the native dimeric myoglobin with ferricyanide oxidizes the haem and converts the myoglobin partly to monomer and partly to varying amounts of dimer and higher molecular weight polymer. 3. Ferricyanide-oxidized myoglobin contains no sulphydryl groups reactive to DTNB or NEM. 4. If the ferricyanide oxidation is carried out in the presence of cyanide, the resulting cyanmetmyoglobin is dimeric with x-2 reactive sulphydryl groups per haem, and can be cleaved to monomer by reaction with PMB. 5. Reassembly experiments with variously modified subunits imply a conformational difference between oxy-, carbonmonoxy-, and cyanmetmyoglobin on the one hand and _ metmyoglobin on the other. 6. Tbe kinetics of myoglobin monomerization have been followed using rapidresponse Sephadex gel techniques.

MYOGLOBIN from the gastropod mollusc Burycon canaliculutum, purified and partially characterized by Read (x966), has been

su~~u~tly shown to be a true dimer, dissociating into identical subunits on reaction with p-hydroxymercuribenzoate (P-1. Reaction with PMB also leads to rapid oxidation of the haem in myogiobin from Busyconcanuliculatum (Johnson, Koppenhe&r, and Read, 1971) and the gastropods Littwina littorea (Koppenheffer and Read, I97 I) and Fusitriton oregwuwis (Terwilliger and Read, 1970). On the other hand, myoglobin from another gastropod, the waved whelk Buccinum usdatum, has been shown (Terwilliger and Read, 1969) to behave similarly to human haem~lobin (Tyuma, Benesch, and Benesch, 1966) in that, although

cleaved

to subunits

by

reaction

with PMB, it does not undergo concomitant haem oxidation. In this work the relation of sulphyd~l blockage and haem oxidation state to the maintenance of the native dimeric condition of B~isyconcu~~~~u~rn myoglobin has been determined. MATERIALS AND METHODS Bzqcon codiculatum L. was obtained from the

Marine Biological Laboratories, Woods Hole, Mass., and kept in holding tanks at the New England Aquarium until needed. The radular muscles were excised and ground in a mortar with acid-washed sea-sand and 0.1 M sodium phosphate, pH 7.4. The uzide extract was fractionated with ammonium sulphate, and the deep red precipitate redissolved in the pH 7-4 extraction buffer. The myoglobin was then chromatographed on a column of Sephadex G-75

JOHNSONAND READ

294

(su~r6ne) in equilibrium with the o* I M sodium phosphate, pH 7.4 buffer. All column chromatography was performed at about 4” C. PREPARATION OF DERIVATIVES Reaction with Ferricpnide Myoglobin purified as above was reacted with a five- to tenfold molar excess of ferricyanide to haem, and the reaction monitored by following the disappearance of the a absorption peak. After I hour of reaction at room temperature (about 20’ C.) the reaction mixture was chromatoerauhed on a column of Sevhadex G-75 in 0.1 M &d&m phosphate, pH 7.4.. Cy~metmyogIobin was prepared in the same way, except that a tenfold molar excess of potassium cyanide to haem was added to the myoglobin solution prior to the addition of ferricyanide. ,”

Reactionwith FM3 Stock solutions of 25 mM PMB were prepared as previously described (Johnson and others, I g7 I). For preparative reactions, aliquots of the stock PMB solution were added to the myoglobin in 0.1 M sodium phosphate, pH 7.4, at intervals of 1-2 minutes until preselected ratios of PMB to haem were attained. The reaction mixture was subsequently applied to a column of Sephadex G-75 in equilibrium with the pH 7.4 buffer. Analytical PMB titrations were performed accordii to Benesch and Benesch (~$52). Titrations were performed at room tem~rature, and followed s~t~photometricaily at 250 nm. Reaction with 5,5’ ~~thio(b~-2-~itrobenzoic acid) (DTJVB) DTNB was reacted with myoglobin essentially according to the method of Ellman (I 959). For analytical determinations the protein blank and reaction cuvettes contained identical 2-ml. aliquots of myoglobin in O-I M sodium phosphate, pH 7.4. Reaction was initiated by adding o-x ml. of 0.01 M DTNB in O-I M sodium phosphate, pH 7.4, to both the reaction cuvette and to a reagent blank, which contained only buffer. Corrections were made for the change in optical density of the reaction cuvette occasioned by dilution with the reagent, and for the continuous, slow increase in absorbance of the reagent blank. The extent of DTNB reaction was then calculated using a mM extinction coefficient for the DTNB reaction product of I 3.6 at 412 nm. For reactions in which it was desired to-test for PMB-reactive sulphydryls after reaction with DTNB, it was necessary to use only a threefold molar ratio of DTNB to haem, otherwise the absorption at 250 nm. would be too great for accurate titration with PMB. Reaction Kinetics with FMB and DTNB Since the myoglobin absorbs strongly at wavelengths used to follow the reaction with PMB and

ht. J. Biochem.

DTNB, the reacting solution was read against an otherwise identical blank containing only myoglobin in the buffer. Myoglobii concentration was determined using a mM extinction coefficient at 540 nm. of 14 for the carbonmonoxy form and 13 for the oxy form (calculated by converting a sample of oxymyoglobin to carbonmonoxymyoglobin, for which the extinction coefficient was known) (Johnson and others, 1971). Reaction was initiated by adding 0.1 ml. or less of reagent to 2.0 ml. of myoglobin
‘97’2

2

SH BLOCKAGEAND HAEM OXIDATIONOF Mb DIMERS

fiH 7.4, was run into a column of Sephadex G-25 previously equilibrated with 0.1 M sodium phosphate, pH 7.4. The small volume of solution containina PMB-derivatives of mvoelobin was then added”and elution performed &ih 0.1 M sodium phosphate, pH 7.4. This sequence of additions ensures that the myoglobin passes through a zone in the column containing 2-mercaptoethanol where the PMB moiety is removed from the myoglobin. The myoglobin emerges well separated from the n-mercaptoethanol. Removal of the PMB causes the monomeric subunits of the myoglobin to reassemble into the dimer. When it was desired to reduce the oxidized haem of the myoalobin undercroinn reassemblv. the 0.1 M sodium phosphate co%_tmGbuffer, pH.7.4, was also made 0.02 M in formamidine sulnhinic acid (FASA) (Shashoua, 1964). Buffer flow-rate was adjusted so that the (approximately) I .g x go cm. column took about 40 hours to deliver the reassembled ferromyoglobin dimer. RAPID AND CONTINUOUS DETERMINATION OF MONOMER:DIMERPROPORTIONS IN MYOGLOBIN REACTING WITHPMB AND DTNB Conventional column gel filtration is far too slow an assay method with which to follow the time course of monomer formation on reaction of Busycon canaliculatum myoglobin with sulphydrylattacking reagents, and thus cannot be used to discover whether monomerization follows or precedes haem oxidation. An alternative, much more rapid procedure, which is a special case of the more general column scanning technique explored by Brumbaugh and Ackers ( I g68), involves running the reacting myoglobin solution through a small ‘ sampling ’ volume of Sephadex gel packed in a flow-cell. The light absorbance of the flow-cell will depend on the (constant) absorbance of the Sephadex gel itself. the absorbance of solution flowing around the gel beads, and the absorbance of solutron inside the nel beads. Ecmilibration of the flow-cell ael with”a solution cdntaining light-absorbing sol;te molecules will darken the gel to an extent which depends on the proportion of the gel-bead volume into which the light-absorbing solute can permeate. Thus, with a proper choice of Sephadex gel, monomeric mvoalobin will r&e a greater contribution to the lrght absorpiion of the flow-cell assemblv than will dimeric mvodobin. if the haem concentrations are equal. Furthermore, if absorption due to a myoglobin species varies linearly with its concentration, it can be shown that the change in monomer concentration of a reacting solution is also linearly proportional to the resulting change in light absorbance. When a myoglobin solution flowing through the cell has equilibrated with the gel phase, the absorbance, A, will be given by

295

where C, = concentration (haem) of monomeric myoglobin; C, = concentration (haem) of dimerit myoglobin ; E, = extinction coefficient of monomer; E,, = extinction coefficient of dimer; A r_,h = the apparent (constant) absorption of the Sephadex gel; V, = proportion of gel bed occupied by gel beads; V, = proportion of gel bed not occupied by beads; S, = gel filtration partition coefficient for monomer (fraction of V, accessible to monomer); S, = gel filtration coefficient for dimer (fraction of V, accessible to dimer) .

The quantities &,(S,V,+ VJ .and E,,(S~V~+ V,), however, are constants for given gel, protein, buffer, and flow-rate conditions, and so may be represented by k II and k,,, respectively. Making the above substitution, equation (I) becomes

A = C,k,+C,k,+A,.,, . (2) If, in the course of an experiment, the myoglobin is converted from entirely dimer to entirely monomer, k, and k, are easily determined from the absorbance at the beginning and end of the experiment, respectively; A,.,, is determined in the absence of myoglobin. If the haem concentration of the reacting solution being fed into the gel remains constant, C,+& = constant = Q. (3) Rearranging equation (2), and substituting in equation (3), c,

=

A-A.~;--CL, n

=

A-A..,,-

(Q-cm)kd

km

(4)

which in turn gives, on collection of the C, terms, =

c I

A-Qkr-A,,,, k,-k,

*

The percentage of myoglobin which is in the form of monomer is then

Cl&l _x ,oo = A-QkrAw

Q

Qkm-Qkd

“00

where A, = Q kl-+-Al,pk = absorbance >hen all the myoglobin is dimeric, and A, = absorbance when all myoglobin is monomeric. The experimental apparatus is schematically illustrated in Fig. I. Reacting solution is pulled through intramedic polyethylene tubing and the gel bed by a Buchler polystaltic pump attached to the effluent line of the flow-cell. Since polyethyIene tubing by itself is rather non-resilient and subject to cracking, the portion in the polystaltic pump has been reinforced by being pulled in a

296

JOHNSON

tight press-fit through a short section of Tygon tubing. The flow-cell is constructed from an inverted ~-cm. path-length quartz cuvette. A small hole bored near the top (sealed) end allows the attachment of a polyethylene inflow tube by pressfit. The (open) bottom of the cuvette is closed by a combination gel-bed support and effluent tubing fitting made of Lucite. The Lucite block has a lip around its lower end, which acts as a seat for a neoprene O-ring placed between the lip and the end of the cuvette. The O-ring effectively seals the assembly when compressed by several wraps of black vinyl tape taken around the four non-optical surfaces of the cuvette.

Photomulriplicr

f FIG. I .-Schematic

ht. J. Biochcm.

AND READ

on the earliest time at which one can begin to follow changes in the molecular weight distribution of the reacting myoglobins. This initial equilibration takes longer than the response time discussed above because there is ‘ dead volume ’ between the reacting solution reservoir and the top of the gel bed, which is the starting point for determining the response time to changes occurring throughout the solution. A high flow-rate and short connexions with the reservoir have cut the time required for achievement of a steady absorbance trace with freshly introduced non-reacting myoglobin dimer to 2 minutes. Two minutes has thus been taken as the earliest time at which useful information can be obtained on the progress of the monomerization reaction.

A

Polyachylme

tubin;

I

diagram of Sephadex gel flow-cell apparatus.

The upper end of the Lucite block, which is inserted with a close (but not tight) fit several mm. into the cuvette, contains a pyramidal depression covered by a nylon net (cemented in place with Lucite solvent) which serves as the actual gel support. The effluent polyethylene tubing is press-fitted into a small hole which connects with the bottom of the pyramidal depression. Once the gel bed has been equilibrated with the reacting myoglobin solution, the response time required for the system to register a change in the degree of myoglobin aggregation will be approximately the time required for a segment of the reacting solution to pass through the column. Since the gel-bed volume is about 0.8 ml. and the flow-rate is about 1.2 ml. per minute, the response time for measuring a change in the relative proportions of monomer and dimer being fed into the column should be about I minute. The time taken for initial equilibration of the flow-cell with reacting solution places a limitation

There is a possibility that intermediates in the dimer to monomer conversion process may have extinction coefficients which differ from those of the dimeric oxymyoglobin and monomeric metmyoglobin derivatives. Such possible intermediates can be prevented from affecting the accuracy with which monomer : dimer proportions are determined by reading the total flow-cell absorbance at a wavelength where all myoglobin species present in appreciable concentration in the reacting mixture have the same extinction coefficient per haem. Several such isosbestic wavelengths have been determined for the reaction of oxymyoglobin with PMB and DTNB. The one at 53 I nm. has been used since it is the one in a most nearly flat portion of the absorption curve. The linear relation between solute concentration and absorbance, assumed in equation (I), is demonstrated in Fig. 2. Note that dexttan blue 2000, which is excluded from the Sephadex G-75 gel beads, contributes relatively little optical

‘971,

SH BLOCKAGE

2

AND HAEM OXIDATION

density to the gel bed. This is probably because, at the high flow-rates used (about 150 ml. per cm.2 per hour), the very significant back-pressure crushes and deforms the gel beads, so that the ‘ excluded volume ‘) VI, which is the only portion of the gel bed accessible to the dextran blue 2000, is greatly reduced. Note also that the requirement for high flow-rates, resulting in a crushed gel, precludes the use of gel filtration exclusion coefficients determined with the same kind of gel

OF

Mb DIMERS

297

great care be taken to keep the flow-rate constant so that the absorbance due to the gel will not vary. It is desirable to keep the gel-bed volume as small as possible so that the effects on subunit activities occasioned by differential partition of monomeric and dimeric myoglobin into the gel beads will be minimized. However, it was decided not to decrease the width of the gel bed as this would decrease the amount of light reaching the

3-

E N .r

2-

A I IL

I

I

I

I

2

I

3

Abwrbmcc of I cm. of sdution (without Scphdex)

Fm. I.-Linear relation of the absorbance of a h-mm. thick Sephadex G-75 (superfine) gel, equilibrated with flowing solution in the apparatus of Fig. I, versus the absorbance of the equilibrating solution in a standard r-cm. path-length cuvette. Absorbance of the flow-cell was read against blanks of arbitrary absorbance. A, Dextran blue aooo, 0, Sperm whale metmyoglobin, Ej, Buryroncanalicdatum dimeric myoglobin. but at normal flow-rates of around 6 ml. per cm.* per hour. A related consequence of the gel crushing is that relatively little of the gel-&ad volume is available to the myoglobin molecules, so that rather high myoglobin concentrations are required to give satisfactorily large absorbance effects in the Sephadex gel. The apparent absorbance of the gel alone is usually about 1.95 for a +mm. pathlength (reduced from I cm. with a 6-mm. Lucite spacer). A dimeric myoglobin solution with an absorption at 531 nm. of about o-5 for a r-cm. path-length without Sephadex will, after equilibration, increase the absorbance of the gel bed by about 0.05 absorbance unit, the exact value depending on the flow-rate. Conversion of the myoglobin to monomer will increase the absorbance by another 0~02-o~o3 absorbance unit. With such a high baseline and relatively smaI1 absorbance changes, it is obviously necessary that

photomultip~er from the highly light-~tte~ng gel (Shibata, 1959). RESULTS All sulphydryl-attacking reagents tested have proved capable of causing dissociation of the native dimeric myoglobin into its subunits. Reaction with PMB, DTNB, NEM, or iodoacetamide leads to nearly complete monomerization, as determined by subsequent chromatography on Sephadex G-75 (Fig. 3A, B, C, D). Reaction with ethyleneimine (EI) for several hours at room temperature, however, results in only partial monomerization. The remaining dimer from the EI-reacted mixture contains 2 free sulphydxyl groups per haem

298

JOHNSONAND READ

(determined by PMB titration), while the monomerized fraction contains none. Thus the failure of EI to cause complete monomerization is probably due to the sluggishness of the reaction rather than to special properties of E&reacted myoglobin. Reaction of the native mvoglobin with any

Int.

3. Biochem.

was titrated with PMB after being incubated at 25’ C. for IO hours, there was still I reactive group per haem available for reaction with PMB. Since the normal complement of PMB-reactive groups in Busycon canalidatum myoglobin is 2 per haem group, it appears that the DTNB reaction involves

Elution volume (ml.)

FIG. 3.-Sephadex G-75 gel filtration, in 0.1 M sodium phosphate, pH 7.4, of Busycon canaliculatum myoglobin reaction products. A, PMELreacted carbonmonoxymyoglobin, 0.4 mM haem, 5 mM PMB, reacted in 0.1 M sodium phosphate, pH 7.5, for 30 minutes at room temperature. Sample volume 5 ml. 8, Iodoacetamide-reacted oxymyoglobin, sample volume 3 ml. C, DTNELreacted carbonmonoxymyoglobin, 0*028 mM haem, 0.9 mM DTNB, reacted in 0.2 M Tris-HCI, pH 8.0, for 45 minutes at room temperature. Sample volume 8 ml. D, NEM-reacted oxymyoglobin, sample volume 3 ml. Column dimensions, about 1.9 x g7 cm., flow-rate, 8-10 ml. per hour. of these reagents results in disappearance of the a absorption peak (576 in the oxymyoglobin, 569 in the carbonmonoxymyoglobin) in the monomeric products. The kinetics of sulphydryl reaction and associated haem oxidation have been followed for reaction with PMB and DTNB (Fig. 4). Haem oxidation clearly lags somewhat behind blockage of the sulphydryl groups. The reaction with DTNB, monitored at 412 nm., cannot be followed accurately once significant haem oxidation has occurred since the associated shift of the Soret band to lower wavelengths obscures further changes in absorbance owing to possible DTNB reaction. However, it was found that if the DTNB-containing reaction mixture

only I of the 2 sulphydryl groups, and that it was essentially complete after 10 minutes. REACTION WITH FERRICYANIDE In an attempt to see whether haem oxidation by itself is sufficient to cause monomerization, myoglobin was reacted with equimolar or greater concentrations of potassium ferricyanide. It was found that up to a fivefold molar ratio of ferricyanide to haem was required before additional aliquots of ferricyanide failed to reduce the absorption further at the former position of the a peak. Sephadex G-75 chromatography of this oxidized myoglobin gave both monomeric and dimeric components (Fig. 5A), neither of which contained sulphydryl groups reactive

SH BLOCKAGE

AND

HAEM

OXIDATION

OF

Mb

DIMERS

FJC, 4.-Kinetics of the reaction of oxymyoglobin with PMB and DTNB. A, Moles PMB reacted per mole of haem: 0*025 mM haem, CPOCJ mM PUB, 0, Percentage haem oxidized in PMB reaction. a, Moles DTNB reacted per mole .haem: 0.025 m&I haem, 0.5 mM DTNB. A, Percentage hem oxidized in DTNB reaction. Reaction temperature PO%.

Elutioo vdumt (ml.) Fm. 5.-Sephadex G-75 gel filtration, in 0’1 M sodium phosphate, pH 7.4. A, Ferricyanidc-oxidized oxymyoglobin, o-09 m&f haem, 0.35 mM K,Fe(CN),, reacted for I hour at room temperature in 0.1 M sodium phosphate, pH 7.4. B, Myoglobin indicated by bar in A, after reaction with NEM. A mixture containing 0.014 xnM hacm and 0-04 mM NEM was reacted for 45 minutes at room temperature in 0.x M sodium phosphate, pH 6.9, then chromatographed. Cohtmn dimensions 1.9 xg8 cm., flow-rate aml. per huur.

300

JOHNSONANDREAD

towards DTNB or NEM. The dimeric myoglobin of Fig. 5A, when reacted with NEM, remained in the dimeric condition (Fig. 58). Similar ferricyanide oxidation of Busycon canaliculatum myoglobin has sometimes produced an additional, higher molecular weight component which has not been characterized. EFFECT OF CYANIDEION ONTHE METMYOCLOBIN If cyanide is included in the ferricyanide mixture, reaction dimeric cyanmetmyoglobin is the only product. This cyanmetmyoglobin contains I-I ‘5 DTNB-reactive

ht. J. Biochcm.

groups (Fig. 7). The monomeric derivatives all have (unavoidably) oxidized haems. If PMB is simply removed from the subunits with n-mercaptoethanol they aggregate to form dimeric molecules which are completely lacking in reactive sulphydryls (tested with PMB and DTNB), and are completely resistant to cleavage by sulphydryl-attacking reagents. On the other hand, if the haem is reduced with formamidine sulphinic acid (FASA), or the oxidized haem liganded with cyanide during reassembly, the regenerated dimer contains reactive sulphydryl groups and can be cleaved to monomer again by reaction

n

Dextran blue zoo0

S. conoliculatum cyrnmatmyaglobin dlmcr M.W. - 32.ooO

I I Sperm whale metmyoglobin

I I

I

Elurmn

volume

(ml.)

FIG. 6.-Scphadex G-75 gel filtration, ino. M sodiumphosphate,PH 7’4, ofcyamnetmyoglobin reacted with PMB: 0.05 mM haem, 0.5 mM PMB, reacted for 30 minutes in 5 ml. of 0.1 M sodium phosphate, pH 7.4, at room temperature. Column dimensions I ‘Qx 98 cm., flow-rate 8 ml. per hour. groups per haem, depending on the prepara-

tion, and can be cleaved entirely to monomer by reaction with PMB (Fig. 6). Removal of the PMB, using the technique outlined in Methods, results in a dimeric myoglobin which is capable of again being cleaved to monomer by reaction with PMB. REA.%EMBLYOF VARIOUSMONOMERIC DERIVATIVES Various monomeric subunit derivatives of Busycon canaliculatum myoglobin can be reassembled to form dimeric myoglobin by removing PMB from blocked sulphydryl

with PMB reagents.

or other

sulphydryl-attacking

INFLUENCEOF PH ANDIONIC STRENGTHON MOLECULARWEIGHT At a pH of 6.0 or 8-g in 4.0 M NaCl, Burycon canaliculatum oxymyoglobin shows no indication of a decrease in its degree of aggregation of Fig. 8.

in the gel filtration

experiments

The high apparent molecular weight (44,000) at pH 6.0 may be related to the fact that the myoglobin precipitates in the range fll-3 5-6, or may simply be due to the inadequacy of globular proteins as

SH BLOCKAGE AND HAEM OXIDATIONOF Mb DIMERS

1971,2

molecular weight markers at these high ionic strengths. In another experiment, 4 hours of dialysis and subsequent chromatography on Sephadex G-75 of the myoglobin in 0.2 M NaCl, 0.01 M sodium phosphate, pH 6.05, resulted MONOMER

DIMER

etmyoglobin

$;:3-y

PMB

,Metmyoglobin

CN-Metmyoglobin

PM6

- Metmyoglobin

CN-Metmyoglobin

PMB

,Metmyoglobin

30’

0.2 M NaCl buffered with O*OI bf Tris at #H 74 and 843 suffered no conversion to monomer. Overnight dialysis at 4” C. of the myoglobin against 0.005 M sodium phosphate, pH 6.05, produced oxidized myoglobin which gave a complex, multipeak pattern when chromatographed on carboxymethylcellulose. MONOMERIZATIONKINETICS ON REACTION WITH PMB AND DTNB

The curve for monomerization on reaction of native dimeric oxymyoglobin with 2 molecules of PMB per haem, determined with the Sephadex gel flow-cell apparatus, is (within the limits of experimental error) the same as that for the attachment of PMB to the myoglobin (Eg. 9). Haem oxidation, which follows apparent first-order kinetics, is seen to lag considerably behind both monomer formation and PMB reaction.

t

Ihtmyoglobin

FIG. 7.-Diagrammatic

summary of carhonmonoxymyoglobin derivatives relevant to discussion of different cotiormational states. State of aggregation was determined by gel filtration on Sevhadex G-775 in 0.1 M sodium phosphate, bH 7-i. Fractio&-comprising 1~ th& ro kr cent of the total myoglobin c~rnat~aph~ were igaored in preparing the diagram. in conversion of 20 per cent of the myoglobin to monomeric metmyogiobin, the remainder of the myoglobin eluting at the position of normal dimer. Dialysis and subsequent gel filtration of a separate aliquot of the same myoglobin in 0.2 M NaCl, 0.01 A4 sodium phosphate, pH 6.9, gave mostly dinner of normal molecular weight, with less than 5 per cent monomer. Myoglobin dialysed against solutions of

FIU. 8.-Molecular weight determination of oxymyoglobin by gel filtration on Sephadex G-75. 0 @, Bovine serum albumin. 0 n , Bu~ycon cunaliculatummyoglobin. v v, Chymominogen A. A A, Sperm whale metmyoglobin. Data obtained in 40 M NaCl, 0. I M sodium phosphate, pW 6-0, are represented by hollow symbols; data obtained in 40 M NaCl, 0. I M Tris-HQ, pH 8.9,

are represented by solid symbois.

The kinetics are quite different in the case of reaction with DTNB, which under the reaction conditions used reacts with only I of the 2 sulphydryl groups. The DTNB attaches quite rapidly, but both monomerization and haem oxidation of the myoglobin lag far behind (Fig. IO). There is some indication that haem oxidation precedes monomer formation in the later stages of reaction, but since the base-line absorbance

of the Sephadex gel has a tendency to drift slightly, the accuracy with which the proportion of monomer in the solution can be determined diminishes with time. It is thus probably more accurate to say that haem oxidation and monomer formation are nearly simultaneous when a single sulphydryl group is blocked with DTNB.

myoglobin subunits, the myoglobin was reacted with a number of sulphydrylattacking reagents in an attempt to find a differential effect with groups of different charge and size. All the reagents reacted and cleaved the myoglobin into its 2 identical subunits, but in addition caused the conversion of the haem iron to the ferric form. Although this effect is not seen on reacting DEXXJSSION human haemoglobin with sulphydryl blocking reagents (Guidotti and Konigsberg, As part of a study on m~fi~tion of the aggregation properties of 3~~0~ ~a~~~~~a~rn 1964; Tyuma and others, 19661, Bruhori,

0

30

20

FIG. 9.--Kinetics of monomerization of dimeric oxymyoglobin on reaction with PMB. 0, Attachment of PMB-I 00 per cent reaction is z moles of PMB Per mole of haem. A, Percentage of myoglobin in monomeric form. 0, Percentage of haem oxidized. 0.05 mM haem, 0.15 mM PMB, in o-x M sodium phosphate,pH 7.4.

e

Time
Fxc. io .-Kinetics ment of DTNB-100

of monomerization of dimeric oxymyo~lobinon reaction with DTNB. 0, Attach-

per cent is I mole of DTNB per mole of haem. A, Percentage of myogiobin in monomeric form. 0, Percentage of haem oxidized. 0.078 mM haem, ~4 mM DTNB, in w I M sodium phosphate, PH 7.4.

197122

SH BLOCKAGE

AND

HAEM OXIDATION

Taylor, Antonini, and Wyman (1967) have shown that reaction of the p-93 sulphydryl with a variety of blocking agents increases the free energy of haem oxidation. Also, Terwilliger and Read (197 I) have found that dimeric erythrocyte haemoglobin of the holothurian echinoderm Molpadia oolitica, which contains no cysteines, can be cleaved to monomer when oxidized with a large excess of ferricyanide. Because sulphydryl blockage of Busycon canaliculatum leads to haem oxidation, one cannot, on the basis of the simple blocking experiments with B. canaliculatum myoglobin mentioned above, conclude that the subunit association was destroyed as a direct consequence of blocking the cysteines. The question of whether haem oxidation by itself sufficiently alters subunit interactions as to cause dissociation is not resolved by reacting with ferricyanide. The requirement for several times more than a molar equivalent of ferricyanide to haem to achieve complete oxidation of the haem group indicates that other groups are also being modified. Although the resulting product contains monomeric subunits, interpretation is complicated by the presence of dimeric and higher polymeric fractions. Since none of these fractions contains reactive sulphydryl groups, the monomer produced through ferricyanide reaction might have resulted from ferricyanide oxidation of the protein’s free sulphydryl groups (Tyuma and others, I 966) rather than from alterations directly caused by changes in haem oxidation state. This uncertainty cannot be resolved by protectively blocking the sulphydryl groups during haem oxidation, for this is equivalent to the experiment in which the cysteines were reacted with PMB (the haem being thereby unavoidably oxidized) and the PMB subsequently removed, with the formation of an oxidized haem dimer lacking reactive sulphydryl groups. An oblique approach to the problem presented itself with the observation (Johnson and others, 1971) that cyanide ions stabilized the dimer during and after haem oxidation with ferricyanide. The resulting cyanmetmyoglobin dimer has I reactive sulphydryl

OF

Mb DIMERS

303

group with respect to DTNB, and, depending on the preparation, 1-2 reactive sulphydryl groups with respect to PMB. An oxidized haem iron, therefore, is not in itself a sufficient condition for monomerization of the dimer or alteration in the availability to reagents of the cysteine side groups. Furthermore, since the cyanmetmyoglobin dimer is split cleanly into subunits upon reaction with PMB, cyanide stabilization of the dimer cannot be due to covalent bonds between subunits which have somehow been induced by the presence of cyanide. It seems likely that the cyanide exerts its effect as a ligand of the haem, although other sites of binding and modes of protective activity cannot be ruled out, especially since the protective effect seems to require more than I cyanide ion per haem group, even with well purified (ammonium sulphate fractionation, Sephadex G-75 chromatography) myoglobin. The splitting into subunits of the cyanmetmyoglobin dimer by reaction with PMB probably results directly from the introduction of PMB at the cysteine residues, rather than from secondary effects associated with changing the haem Iigand, since the cyanide ions seem to remain attached to the myoglobin even after monomerization. That the cyanide remains attached is indicated by the fact that removal of PMB from the PMBcleaved cyanmetmyoglobin monomer results in the formation of a cleavable dimer with reactive sulphydryls, typical of molecules which have been reassembled in the presence of excess added cyanide ; this is in distinct contrast to the unreactive dimers formed from monomeric PMB derivatives in the absence of cyanide or haem reduction (Fig. 7). There is evidence, in terms of sulphydryl reactivity, for a conformational difference in the isolated subunits which depends on the oxidation and ligand state of the haem. When PMB is removed from monomeric PMB derivatives the subunits aggregate to form oxidized haem dimers (with less than IO per cent residual monomer) which resemble the oxidized dimeric myoglobin produced by reaction of the native oxy- or carbonmonoxy-myoglobin with ferricyanide

3%

JOHNSON

to the extent that both kinds of oxidized haem dimers lack reactive sulphyd~ls. Whether the sulphydryls are oxidized, covalently bonded, or simply sterically hidden in these molecules is not known, although tentative results from reaction with DTNB in 8 M urea indicate the absence of reactive sulphyd~l groups* If, instead of merely removing the PM3 from the oxidized haem derivatives, the haem is also reduced, the resulting dimeric, reduced-haem myoglobin appears to be entirely normal, not only in its molecular weight and absorption maxima, but also in its sulphydryl reactivity and ability to be cleaved to monomer (with associated haem oxidation) upon reaction with PMB. The haem-reducing agent (FASA) is not included in the actual PMBremoving Imercaptoethanol buffer segment, but in the column equilibration and eluting buffer. Thus, although at the time of PMB removal the haem is still in its oxidized state, the myoglobin is apparently not immediately locked into the ‘ unreactive sulphydryl ’ form typical of the oxidized haem dimer, since reduction of the haem iron with FASA farther along in the column is sufficient to preserve sulphydry1 reactivity. The essential point to be noted in all these manipulations is that haem reduction subsequent to PMB removal, or the presence of cyanide ion during PMB removal, is sufficient to cause the subunits to assume a form which protects the sulphydryls from airoxidation or from being sequestered in the interior of the molecule, and permits the formation of apparently normal dimeric aggregates. This is in contrast to the sulphydryl-less dimers formed from oxidized haem subunits. Tyuma and others (x966) have found that although human haemoglobin A could be oxidized to stable ferrihaemoglobin by ferricyanide with complete retention of free sulphydryl groups, oxidation of the isolated a and p subunits with ferricyanide caused much denaturation and precipitation. The inclusion of cyanide in the reaction mixture stabilized the product and prevented precipitation, but unless the reaction conditions

AND

READ

ht. J. 3&?&m.

were carefully chosen, there was still some oxidation of the cysteine side-chains. It may be that cyanide exerts its stabilizing effect by ensuring that a conformation characteristic of globin with properly bound haem is maintained at all times, since it has been found that cyanide blocks haem exchange in ferrihaem~lobin (Bunn and Jandl, x968; Jahaverian and Beychock, I 968). The sequence of events involved in the monomerization and haem oxidation resulting from PMB reaction of the native dimeric oxymyoglobin have been clearly established (Eg. 9). It is obvious that the breaking apart into subunits is nearly simultaneous with the attachment of PMB, and clearly precedes the haem iron oxidation. Thus there is no question in this case about haem oxidation being the cause of monomerization; one must consider instead whether the haem oxidation results from conformational change of the subunit on monomerization, from loss of the steric protection offered to the haem of one subunit by its partner subunit, or else from some effect of PMB attachment not related to monomerization. The haem oxidation rate (Fig. g) approximates first-order kinetics, but since the PMB attachment and monomerization are both more than go per cent complete within 3 minutes, the later portions of the haem oxidation data give no further information about the possible necessity for monomerization prior to haem oxidation, while data earlier than 3 minutes lack sufficient resolution to be able to tell if the reaction differed significantly from first-order kinetics in the early portion. The sequence of changes occurring in oxymyoglobin reacted with DTNB (Fig. IO) indicates that blockage of only I sulphydryl group per haem is not sufhcient for immediate monomerization, and that some later ratedetermining alteration is involved. Because of slow base-line variations with time, the monomerization kinetics shown in Fig. IO are sufficiently uncertainso that one cannot say whether the haem oxidation follows or precedes the cleavage into subunits. However, since there is still one sulphydryl group which can react with PMB even after incubating

‘97’,2 the myoglobin

SH BLOCKAGE

AND

HAEM

overnight with DTNB, it is certain that the subunit formation is not dependent on reaction of the second cysteine with DTNB. The curves for monomer formation and haem oxidation are so close together that it is extremely likely that either both alterations result from a common, rate-limiting change in the protein initiated by the reaction with DTNB, or that one of the two monitored alterations is a consequence of the other. It seems probable that the haem oxidation has some effect on monome~zation, for in vertebrate haemoglobins different sulphyd~lattacking reagents often exhibit differing degrees of effect on aggregation (for instance, Rosemeyer and Huehns, I 967). The ultimate effect of all such reagents tested on Busycon canaliculatum myoglobin, however, has been complete dissociation. Since it is established that at least one of the reagents (DTNB) cannot cause immediate monomerization by blocking one sulphydryl group, it seems possible that the peculiar feature of undergoing haem oxidation on blockage of the sulphydryl groups might well account for the uniform dissociating effect of all these reagents on Bus~conca~~~~~~ myoglobin. It is not known why PMB reaets with both sulphydryl groups while DTNB reacts with only one. Perhaps one of the cysteine residues is more sheltered from the rather bulky DTNB, although the unreactive residue is probably not actually deep in the contact interface between subunits, otherwise it would not be reactive with PMB. On the other hand, if attachment of one PMB per haem were sufficient for monomerization, this would serve to expose the other cysteine and lead to its reaction. Limitation of the PMB to I molecule per haem, however, results in the production of about 50 per cent monomer, much lower than would be expected if I PMB per haem were sufficient for monomerization. A rapid equilibrium between dimer and monomer which might allow PMB to react with a sulphydryl group in the subunit interface should also allow reaction of DTNB, but even after overnight incubation with excess DTNB there is still only I unreacted sulphydryl.

OXIDATION

OF Mb DIMERS

305

Finally, it should be noted that the cleavage to monomeric subunits cannot be due to smaI1 changes in #H or ionic strength associated with addition of reagents or reaction conditions. The myoglobin retains at least a dimeric molecular weight from pH 6 to PH 8.9, and from NaCl concentrations of o-2 M to 4-0 M. This stability to high salt concentration also suggests, since the subunits are known not ;o’ be. linked by covaient bonds, that ionic bonds are relatively unimportant in association of the subunits. ACKNOWLEDGEMENT

This

work

was supported

Research Grant Number HEi

U.S.P.H.S. (HEM).

by

REFERENCES BENFXH., R., and BENESCH,R. E. (rg6n), ‘ Determination of -SH groups in proteins ‘, Me& biochem. Analysis, IO, 618-622. BRUMBAUGH, E. E., and Aormns, G. K. (rg68), ‘ Molecular sieve studies of interacting protein svstems. III. Measurement of solute nartitioning by direct t&a-violet scanning of gel colmuns ‘, j. bid i&m., w 6315-6324. BRUNORI, M., TAYMR, J. F., ANTONINI,E., and WYUN, J. (r967), ‘ Studies on oxidationreduction potentials of heme proteins. 6. Human Hb treated with various sulfhydryl reagents ‘, 3. biol. Chem., 242,22g5-2po. . BUNN, H. F., and JANDL,J. H. (rg68), ‘Exchange of heme among hemoglobins and between hemoglobin and albumin ‘, j. biol. Chem., w 465ELTON, G. L. (tg5g), ’ Tissue sulfhydryl groups ‘, Archs Bioehem. Biophys., 82, 70-77. GUIDOTTI. G., and KONI~~BERG.W. (1964). The ~ character&ion of modified human hemoglobin. 1. Reaction with iodoacetamide, and ~~~~eirnide ‘, 3. bid. Chem., 233, 1474”

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JAHA&UAN, K., and BEYCHOK,S. (x968), ‘ Subunit interactions in the conformational change of horse apohemoglobin on binding of hemin ‘, J. mol. Biol., 37, I--I I. JOHNSON, J. P., KOPPENHEFFER,T. L., and READ, K. R. H. (rg7r), ‘ Identical subunits of the dimeric myoglobin of the gastropod mollusc Busycon canaliculatum L.‘, Int. 3. Biochem., a, 65KC%ZNHEFF~R, T. L., and F&AD, K. R. H. ‘ The myoglobin of the gastropod (r97t)t mollusc Littotimz l&area L.‘, Znt.3. B&&em., I, 457-464.

306 READ, K. R. H.

JOIiNSONAND

(1&6), ‘ The characterization of radular muscle’ myo&obins from the gastropod molh.tsc BusvconcaaalicalatamL.‘., Comb. . Biochem. Physiol., x7,.&5-390. ROSEMEYER,M. A., and HUEHNS,E. R. (rg67), ‘ On the mechanism of the dissociation of haemoglobin ‘, 3. mol. Viol., 25, 253-273. SHASHOUA, V. E. (rg64), ‘ Formamidine sulfinic acid as a biochemical reducing agent ‘, Biochemis&y, b 171~1720. SHIBA~A,-K. 1 ig59j, ‘ Spectrophotometry of translucent biological materials ‘, Me& biochem. A?U$J&, 7, 77-I&& TERWILLICSER, R. C., and Ram, K. R. H. (rg6g), ‘ Quaternary structure of the raduiar muscle myoglobin of the gastropod mollusc Bvccinum undatum L.‘, Corn?. B&hem. Physioi., 31, 55-64. TERWILLX~ZR,R. C., and READ, K. R. H. ( rg7o), ’ The radular muscle myoglobins of the gastropod molluscs, Acmaea teshcdinalis scutum Esch-

READ

schoitz, ffatiotis kamtschztkana Jones, Teguio funcbralis Adams., F&t&n orcgownsisRedfield, and Thais lamellosaGmelin ‘, Int. j. Biochem., I, 445-450. TERWILLIGER,R. C., and READ, K. R. H. ( 197 I ), ‘ Purification and properties of erythrocyte hemoglobin from the holothurian echinoderm Mol&iia oolitica ‘, in the press. TYUMA, I., BENBCH, R. E., and BENESCI-I,R. (x966), ‘ The preparation and properties of the isolated a and fl subunits of hemoglobin A ‘, ~~~~t~, % og57---2g62.

xky Word Index: Heme oxidation, myogiobin, aggregation of protein, sulphydryl blockage, Buycon canaliculatum, ~hydroxymercuribenxoate, Nethymaleimide, kinetics of myoglobin monomerixation, Sephadex.