A novel reaction of hemoglobin in invertebrate tissues II. Observations on molluscan muscle

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BIOCHIMICA ET BIOPHYSICA ACTA

BBA 45255 A NOVEL REACTION OF HEMOGLOBIN

IN I N V E R T E B R A T E

TISSUES

I I . O B S E R V A T I O N S ON M O L L U S C A N M U S C L E BEATRICE A. WITTENBERG, J. B. WITTENBERG, S. STOLZBERG AND E . V A L E N S T E I N

The Department of Physiology, Albert Einstein College of Medicine, New York, N.Y. (U.S.A .) (Received March i6th, 1965)

SUMMARY I. S p e c t r a of slices of r e d muscle from t h e m a r i n e g a s t r o p o d molluscs Aplysia californica a n d Busycon caniculatum were e x a m i n e d in a recording s p e c t r o p h o t o meter. The s p e c t r u m seen is t h a t of hemoglobin with negligible c o n t r i b u t i o n from o t h e r pigments. 2. Muscle slices when sealed in a small c h a m b e r e x h a u s t the 02 dissolved in their substance. U n d e r these conditions, the h e m o g l o b i n of B u s y c o n muscle a n d of muscle from one n a t u r a l p o p u l a t i o n of A p l y s i a is c o n v e r t e d to a form e x h i b i t i n g an a b s o r p t i o n s p e c t r u m c h a r a c t e r i z e d b y m a x i m a at 422, 548, a n d 570 m/,. The reaction is reversible, since the o x y h e m o g l o b i n s p e c t r u m r e a p p e a r s when the tissue is r e t u r n e d to 02. 3. Muscle from A p l y s i a of a different n a t u r a l p o p u l a t i o n , u n d e r anaerobic conditions, d i s p l a y s the s p e c t r u m of d e o x y h e m o g l o b i n . A f t e r exposure to N a C N or N a N a slices of this same muscle u n d e r anaerobic conditions d i s p l a y the s p e c t r u m c h a r a c t e r i z e d b y a b s o r p t i o n m a x i m a at 422, 548, a n d 57o m/,. The r e a c t i o n is rev e r s e d b y a d m i t t i n g Oz. 4. The suggestion is a d v a n c e d t h a t the d e r i v a t i v e of h e m o g l o b i n c h a r a c t e r i z e d b y an a b s o r p t i o n m a x i m u m at 422 m/~ is involved, possibly as an electron carrier, in c y a n i d e - i n s e n s i t i v e 0 2 c o n s u m p t i o n .

INTRODUCTION W e h a v e r e c e n t l y r e p o r t e d t h a t h e m o g l o b i n p r e s e n t in t h e nerves of an annelid, A p h r o d i t e , a n d of a mollusc, A p l y s i a , is c o n v e r t e d to forms e x h i b i t i n g u n f a m i l i a r a b s o r p t i o n s p e c t r a when the tissues are allowed to e x h a u s t the Oz dissolved in their s u b s t a n c e 1. The r e a c t i o n s are reversible. The s p e c t r u m of o x y h e m o g l o b i n r e a p p e a r s when the tissue is r e t u r n e d to 0 2. The chemical n a t u r e of the form of h e m o g l o b i n e x h i b i t i n g this s p e c t r u m is n o t known, a l t h o u g h several possibilities including a higher o x i d a t i o n s t a t e of hemoglobin, have been considered 1. F o r convenience we shall refer to this form of h e m o g l o b i n as t h e 4 2 2 - d e r i v a t i v e of hemoglobin since it is c h a r a c t e r i z e d b y a Soret a b s o r p t i o n m a x i m u m at 422 m/z. W e now e x t e n d these o b s e r v a t i o n s to the muscles of two m a r i n e g a s t r o p o d molluscs: Aplysia californica a n d Busycon caniculatum, a n d in a d d i t i o n , show t h a t f o r m a t i o n of the 422-derivative can be i n d u c e d b y t r e a t i n g the tissue w i t h N a N a or NaCN.

Biochim. Biophys. Acta, lO9 (I965) 530-535

HEMOGLOBIN IN MOLLUSCAN MUSCLE

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MATERIALS AND METHODS

Animals Aplysia californica were purchased from Dr. R. FAY, Pacific Biomarine Supply Co., Venice, Calif.

Busycon caniculatum were obtained from the Marine Biological Laboratory, Woods Hole, Mass. Cats were anesthetized with nembutal and exsanguinated from the neck vessels. Tissue slices Muscle slices were cut with a STADIE-RIGGS microtome 2 (A. H. Thomas Co., Philadelphia, Pa.), and were kept in cold sea water or Locke's solution before use. Slices were cut from the buccal muscle mass of Aplysia and from the odontophore muscles attached to the radula of Busycon.

A plysia muscle hemoglobin Aplysia muscle hemoglobin was prepared by the method of ROSsI-FANELLI AND ANTONI~I~, and oxidised to the ferric state with potassium ferricyanide.

Spectra Spectra were determined in a Cary Model-ii recording spectrophotometer. To examine muscle slices anaerobically, the slice in a drop of sea water or Locke's solution was placed on an opal glass plate 4, ringed with grease (Apiezon-T), and covered with a clear coverglass which was held in place b y the grease. A mask, cut from adherent black tape, limited the light so that all the light passed through the specimen. The preparation was positioned in the sample beam of the spectrophotometer with the opal glass between the specimen and the phototube and as close as possible to the phototube. A piece of opal glass bearing a similar mask was placed in the reference beam. To examine muscle slices aerobically, they were mounted between two metal screens and placed in the sample beam. A piece of masked opal glass between the specimen and the phototube improved the resolution slightly. The entire sample compartment was flushed with wet air or wet 0 2. A piece of masked opal glass was placed in the reference beam. RESULTS The buccal muscles of Aplysia and the radula muscles of Busycon are dark red. The color is due to hemoglobin which is present in 0.3 mM concentration 3 (this work), or about 5 % of the dry weight of the tissue. The triturating stomach muscles of Aplysia are somewhat less red. Hemoglobin has been purified from Aplysia muscle 3 and some of its properties studied1,3,5: in particular the molecular weight is about 17 ooo (ref. 5). Busycon hemoglobin is easily purified 6 but the molecular weight has not been established.

Aplysia Aplysia flom two different natural populations were studied. Both were collected from the same b a y on the Southern California coast and are probably of the

Biochim. Biophys. Acta, lO9 (1965) 530-535

532

B.A. WITTENBERGet al.

same genetic stock. One population, which will be referred to as "intertidal animals", was collected from the intertidal zone where they are exposed to surf and to strong sunlight and where they may be desiccated during low tides. These animals appear to feed on red algae. They are deeply pigmented, reddish to reddish black. The second population was collected from 5-1o m depth and will be referred to as "subtidal animals". Subtidal animals appear to graze on brown algae predominantly, and are more brown in color. They are very much less intensely pigmented than intertidal animals. Although animals from both populations are about the same size, the tissues of subtidal animals appear much less substantial, more watery and actually quite translucent, suggesting that their substance is less. The hemoglobin concentration in the peripheral nerve is less in subtidal than in intertidal animals.

Aplysia muscleforming the 422-derivative of hemoglobin The muscles of Aplysia collected in the intertidal zone, when examined anaerobically, have always (five samples from four individuals) displayed the spectrum of tile 422-derivative of hemoglobin (Fig. I). This spectrum is characterized by a Soret maximum at 422-424 m/~ and by a broad absorption in the visible region with evident but poorly resolved maxima at 548 and approx. 57 ° m/~. It is not different from the spectrum displayed by anaerobic nerves from intertidal Aplysia. The consistent behaviour of muscles of these animals recalls the consistent behaviour (four samples from three individuals) of nerves of animals drawn from the same population, which also exhibit the spectrum of the 422-derivative of hemoglobin in the absence of air 1. The muscle slices prepared from buccal or triturating stomach muscles rapidly exhaust the 02 in their substance and in the sea water of the small chamber in which they are examined. The spectrum of the 422-derivative of hemoglobin frequently appears in less than the time required to mount the slice in the chamber and trace the spectrum, about 5-1o min.

Aplysia muscleforming deoxyhemoglobin Muscles of Aplysia collected from the subtidal zone, examined under anaerobic conditions, consistently display a spectrum characterized by a Soret maximum at 434 m/z and by a single relatively broad symmetric absorption band centered at 560 m/z in the visible region (Fig. I). Tile spectrum is recognized to be that of deoxygenated ferrous hemoglobin. The spectrum of deoxygenated hemoglobin is obtained reproducibly in muscle slices from subtidal animals (eight slices from eight individuals). Consumption of 0 2 by the slice sealed in a chamber is rapid.

Aplysia muscle exposed to cyanide and azide Muscle slices prepared from the buccal muscle of subtidal Aplysia were studied in the presence of o.I M NaN a or o.oi M NaCN. (It will be recalled that cyanide diffuses rapidly through tissue as undissociated HCN.) A separate control slice from each animal was examined in sea water to verify the formation of deoxyhemoglobin in the absence of cyanide or azide. Slices were exposed for 5 rain to o.oi M NaCN or o.i M NaNa in sea water and then were sealed in a closed chamber. 02 consumption, judged by the rate of disappearance of the oxyhemoglobin spectrum, was sluggish, Biochim. Biophys. Acts, lO9 (1965) 530-535

HEMOGLOBIN IN MOLLUSCAN MUSCLE

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frequently taking about 15-25 rain. This slow, cyanide-insensitive 0 2 consumption persisted for a long time as could be shown easily by opening the chamber to admit air, and once again following the rate of oxyhemoglobin disappearance. In every slice examined the spectrum of the 422-derivative of hemoglobin appeared as soon as the oxyhemoglobin spectrum had faded away (Fig. 2). Once formed, the spectrum of the 422-derivative of hemoglobin persisted unchanged for more than 30 min. 0.7

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Fig. i. Spectra of anaerobic A p l y s i a muscle. Solid line: m u s c l e f r o m a n i m a l collected in t h e intertidal z o n e e x h i b i t i n g t h e f o r m of h e m o g l o b i n c h a r a c t e r i z e d b y an a b s o r p t i o n m a x i m u m at 422 mff (for clarity this c u r v e is displaced u p w a r d o.I a b s o r b a n c e u n i t in t h e visible region). D o t t e d line: m u s c l e f r o m a n i m a l collected f r o m t h e s u b t i d a l z o n e e x h i b i t i n g t h e s p e c t r u m of d e o x y h e m o g l o b i n . T h e o r d i n a t e scale refers to t h e visible spectral region. T h e a b s o r b a n c e in t h e n e a r - u l t r a v i o l e t region is reduced 5-fold. Fig. 2. S p e c t r u m of A p l y s i a m u s c l e in t h e presence of o . o i M ~ a C N . The m u s c l e is f r o m an a n i m a l collected f r o m t h e s u b t i d a l zone. A separate slice displayed t h e s p e c t r u m of d e o x y g e n a t e d h e m o globin u n d e r a n a e r o b i c c o n d i t i o n s in t h e absence of cyanide. T h e o r d i n a t e scale refers to t h e visible spectral region. T h e a b s o r b a n c e in t h e n e a r - u l t r a v i o l e t region is reduced 5-fold.

These slices when returned to air or 0 2 exhibited a somewhat distorted spectrum of oxyhemoglobin. We do not know whether the distortion is due to changes in the hemoglobin or in the opacity of the muscle. These slices made anaerobic a second time again showed the spectrum of the 422-derivative of hemoglobin. The spectrum of the 422-derivative of hemoglobin seen in cyanide- or azidetreated muscle (Fig. 2) was not distinguishable from the spectrum of the 422-derivative of hemoglobin in untreated anaerobic muscle of intertidal animals (Fig. I) or in Aplysia nerve.

Busycon muscle Slices of the radula muscle of several individual Busycon examined under conditions where the 02 was exhausted displayed the spectrum of the 422-derivative of hemoglobin. This spectrum is not distinguishable from the spectrum of the 422derivative in Aplysia muscle or nerve. Busycon nerve contains a hemoglobin very similar to the muscle hemoglobin (BuRcH AND STRITTMATTER,private communication). Unfortunately other pigments Biochim. Biophys. Acta, lO9 (1965) 530-535

534 mask the hemoglobin spectrum in the intact n e r v e unpublished results).

B.A. WITTENBERGet al. (BROWN AND WITTENBERG,

Spectra of cat soleus muscle MILLIKAN 7, in his classic study of the reactions of myoglobin in vivo demon-

strated that the amount of blood present at any one time in the soleus of the cat was small compared to the amount of myoglobin. Using the very sensitive Hartridge reversion spectroscope he showed that the spectrum of the living muscle in situ with the blood supply intact, was due almost entirely to myoglobin. Slices of soleus muscle consumed 0 2 very rapidly. The spectrum of the anaerobic tissue could be interpreted as the sum of the spectra of deoxygenated myoglobin and lesser amounts of reduced cytochromes. In the presence of cyanide or azide 0 2 consumption was abolished, and the spectrum of oxyhemoglobin persisted for a very long time in slices sealed in a closed chamber. The spectrum of the 422-derivative of hemoglobin was not seen in cat muscle. No sluggish cyanide-insensitive respiration was detected in this tissue. DISCUSSION Cyanide and azide most probably inhibit the respiration of Aplysia muscle by combination with cytochrome oxidase (EC 1.9.3.1 ) (cytochrome as). GHIRETTI et al. s have used difference spectra of particulate preparations from Aplysia muscle to establish the presence in this muscle of a complete cytochrome system composed of the five components: cytochrome a, b, c, Cl, and a s. Cytochrome a s was identified as the terminal oxidase. The 02 acceptor of the cyanide-insensitive respiration has not been identified. The possibility that it is a flavoprotein m a y be considered since GHIRETTI et al. s detected relatively large amounts of flavoprotein in their particulate preparation. If the action of azide or cyanide is solely to block cytochrome oxidase, then we m a y consider the possibility that the 422-derivative of hemoglobin which appears in cyanide- or azide-treated muscle m a y be involved, possibly as an electron carrier, in the cyanide-insensitive respiration. The spectra of the 422-derivative of hemoglobin seen in Aplysia and Busycon muscle are indistinguishable. The structure reflected in the absorption spectrum therefore must be similar in the two proteins. The two hemoglobins differ, however, in m a n y ways including the spectra of the deoxygenated form 9, the spectra of their ferric derivatives a and of the higher oxidation states produced b y H20 2 oxidation (WITTENBERG AND WITTENBERG, unpublished results). Earlier 1 we considered the possibility that the 422-derivative of hemoglobin m a y not be a single compound but any one of several possible mixtures. The finding of the 422-derivative in Busycon muscle, makes improbable the suggestion that the 422-compound is a mixture of ferric hemoglobin with other forms, since purified Busycon hemoglobin does not form a native ferric derivative but is irreversibly converted to the ferric hemochromogen (WITTENBERGAND WITTENBERG,unpublished results). It is necessary to rule out the trivial possibility that the spectrum seen in azide- or cyanide-treated muscle is in reality ferric azide hemoglobin or ferric cyanide Biochim. Biophys. Acta, lO9 (1965) 53o-535

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hemoglobin. Ferric cyanide hemoglobin is indeed formed in Aplysia muscle slices which have stood in air at room temperature for several hours before exposure to cyanide. The spectrum of the ferric cyanide hemoglobin m a y be distinguished from t h a t of the 422-derivative, because the visible m a x i m u m of the former is at 54 ° m/~ and symmetric, that of the latter at 548 m/~ and asymmetric because of the contribution from the 57o-m/~ band. The differences, particularly in the Soret region, however, are small. The spectra of ferric azide Aplysia hemoglobin is very similar to ferric azide horse-blood hemoglobin 1°, and is totally different from that of the 422derivative. No confusion is possible. ACKNOWLEDGEMENTS

I t is a pleasure to acknowledge the skilled assistance of Miss L. KUClNSI
Biochim. Biophys. Acta, lO9 (1965) 530-535