The combination of formyl-substituted haemins with nitrogenous ligands

487

BIOCHIMICA ET BIOPHYSICA ACTA

~BA 25304 T H E COMBINATION OF F O R M Y L - S U B S T I T U T E D HAEMINS W I T H NITROGENOUS LIGANDS R. L E N I B E R G AND A. V E L I N S

Institute of Medical Research, The Royal North Shore Hospital, Sydney (Australia) (Received December 3rd, 1964)

SUMMARY

In contrast to protohaemin which dissolves in purified pyridine to give pyridine ferriprotohaemochrome, formyl-substituted haemins (haemin a, monoformyldeuterohaemin and chlorocruorohaemin) combine with non-aqueous pyridine to form ferrohaemochromes. Their solutions in fl-picoline and diethylamine are also largely ferrohaemochromes, whereas those in ~-picoline, 2,6-1utidine, 2,4(5)-lutidine and triethylamine are ferrihaemochromes or mixtures of ferrihaemochromes with free haematins. These differences between ligands indicate that the steric factor is of greater significance than the basicity of the ligand. The absorption spectra of the ferrohaemochromes in water-free solvents without dithionite differ somewhat from those in dilute aqueous or aqueous-alkaline solutions in the presence of dithionite, particularly in the position of the ~-band; and, with regard to haem a but not the other formyldeuterohaems, in the presence of a weak fl-band in the non-aqueous solutions. On dilution of the pyridine solution with water, the ferr0haemochromes are gradually autoxidized to ferrihaemocbromes, which cannot be reduced b y the removal of atmospheric oxygen: The exception is haem a whose partly autoxidized solutions in 5o-8o % aqueous pyridine are reduced on evacuation; this has been found to be caused b y reducing impurities in the haemin a preparation, not by the removal of reversibly bound oxygen.

INTRODUCTION In a previous paper 1 it has been shown that (ferric) haemin a dissolved in nonaqueous pyridine yields the ferrohaemochrome in the absence of any added reducer, and that the absorption spectrum of felTohaemochrome a in pyridine differs somewhat from that in dilute aqueous pyridine solutions or in 20 % pyridine-o.i N NaOH in the presence of sodium dithionite, which is necessary to prevent autoxidation in these solutions. The observation of CAUGI-IEY AND YORK9' that evacuation restored the ferrohaemochrome spectrum in solutions of partially oxidized haemochrome a in 50-80 % aqueous pyridine was confirmed. The question of whether this was due to the formation of a reversible ferrous--oxygen compound on autoxidation was then left open, but several observations contradicted such an assumption. The rates of Biochim. Biophys. Acta, lO4 (1965) 487-495

488

R. L E M B E R G , A. V E L I N S

autoxidation and reduction on evacuation were small and the absorption spectra of the autoxidized solutions greatly resembled those of ferrihaemochrome a. In the present paper these studies are extended to some other formylhaem compounds (monoformyldeuterohaem and chlorocruorohaem) and to some other nitrogenous ligands. It is shown that the other monoformylhaemins behave like haemin a if dissolved in pyridine, except that the reduction of partly autoxidized haemochrome on evacuation is a specific property of the haemin a preparation. Some of the nitrogenous ligands (/3-picoline, diethylamine) reacted with the haemins in the same way as pyridine, forming ferrohaemochromes, while others formed ferrihaemochromes or mixtures of ferrihaemochromes with free haematins. METHODS AND M A T E R I A L S

Haemin a Haemin a was prepared by reintroduction of iron into porphyrin a prepared as described by MORELL, BARRETT AND CLEZY3.

Monoformyldeuterohaemin dimethyl ester Monoformyldeuterohaemin dimethyl ester was prepared by re-introduction of iron into a monoformyldeuteroporphyrin dimethyl ester of m.p. 26o-268 °, which consists predominantly of the 4-monoformyl isomer with a small admixture of the 2-monoformyl isomer 4. Usually, the ester was used as such as a solution in the organic solvent, but for the study of the spectra of the haemin and haematin in aqueous alkali, the free haematin was first obtained b y saponification of the ester in methylalcoholic K O H at room temperature. The haematin was driven into ether by acidification, the ethereal solution washed with dilute hydrochloric acid and evaporated to dryness. The haemin (free acid) was dissolved in o.I N NaOH. This solution was also used for tile study of the spectra in ammonia or alcohol in sodium hydroxide.

Chlorocruorohaemin dimethyl ester Chlorocruorohaemin dimethyl ester was prepared by oxidation of protoporphyrin dimethyl ester by permanganate ~ and by permanganate plus sodium metaperiodate*.

Ligands Pyridine (British Drug Houses Analar Grade) was purified by distillation over potassium permanganate and finally dried b y distillation over solid potassium hydroxide. A solution of protohaemin in this pyridine showed the ferrihaemochrome spectrum. The picolines and lutidines were B.D.H. laboratory reagents and used as supplied. Diethylamine and triethylamine were redistilled and fractions distilling at the correct temperatures used. 4-Methylimidazole was prepared from the oxalate 8.

Spectra Absorption spectra were measured with a Perkin-Elmer "35o" recording spectrophotometer. Corrections for the wavelength scale were carried out by using * U n p u b l i s h e d o b s e r v a t i o n of t~ARRETT.

Biochim. Biophys. Acta, lO 4 (I965) 4 8 7 - 4 9 5

489

FORMYL-SUBSTITUTED I-IAEMOCHROMES

the ferrocytochrome c m a x i m u m at 55o m/~. The most significant features of the absorption spectra are reported in the Tables I-VI. These give the positions of the a- and y-band maxima, the y/a ratio, the position and relative strength of the minimal extinction for the ferrohaemochromes (but not for the ferrihaemochromes which have only a rather indistinct minimum), and the ratios R 1 and R v The numerators of these ratios are the extinctions at the maximum of the a-band of the respective ferrohaemochromes (R1) and of the y-band (R2) ; the divisors are, for R 1, the absorption of the m a x i m u m of the free haematins (630 m/~ for haematin a, 620 m/~ for the other haematins) and, for Rz, the extinctions at the somewhat arbitrarily chosen y-maxima of the ferfihaemochromes (414 m/~ for ferrihaemochrome a, 404 m/~ for the other ferrihaemochromes). R 2 can be considered as indicating mainly the degree of reduction, although it m a y also depend to some extent on the dissociation of the ferrihaemochrome to the free haematin; R 1 depends on this dissociation to a large extent. RESULTS

Table I shows the absorption spectra of pyridine ferrohaemochromes of haem a, monoformyldeuterohaem and chlorocruorohaem in 20 % pyridine-o.o8 N N a O H Na2S20 4. The spectra of the three ferrohaemochromes are quite similar, except that the ~-maximum of haemochrome a lies 7 m/~, the y-maximum 3 m~ towards longer wavelengths than those of the other two ferrohaemochromes and that, in contrast to the other formylhaemochromes, ferrohaemochrome a has no B-band v,s. The minimum of ferrohaemochrome a lies about io m/~ more towards shorter wavelengths than the first minimum of the other ferrohaemochromes, which is separated from a second minimum at about 530 m~ by the weak B-band at 543 m/,. TABLE I PYRIDINE

FERROHAEMOCHROMES

I N 2 0 ~/o P Y R I D I N E - O . O 8

N

NaOH-Na,S204

in m~. I n parentheses e x t i n c t i o n s relative to a - b a n d m a x i m u m .

Haem

o~-Maximum Minimum

Haem a

588

(0.29-0.46) 431

RI*

R2**

(3.8-5.o) 5.8-I4.5

1.59-I.92

M o n o f o r m y l d e u t e r o h a e m 581

552.5 *** (0.33-0.44) 428.5 (4.8-5.1) 4.4-12.5

1.78-2.o8

Chlorocruorohaem

552.5 *** (0.43)

2.32

581

542

y-Maximum

428

(4.95)

4.4

~/e630 m/z for h a e m a; ea/e620 m/~ for the other two haems. * R2 er/eai4my for h a e m a; ey/e40~ mtz for the other two haems. *** This m i n i m u m is separated b y the weak B-band at 543 m # from a second m i n i m u m at ** R 1 =

approx.

527

m~.

With 4-methylimidazole (1.2-2.5 %) at p H 7-9 a somewhat different haemochrome a spectrum was obtained, with an a-band at 588 m/~, a y-band at 433 m/~, a minimum at 535 m/,, and a weak B-maximum at 516 m/~. The low values of R 1 (2.78) and R2 (1.o4) indicated either incomplete reduction or secondary alteration (c/. ref. I). Biochim. Biophys. Acta, lO 4 (1965) 487-495

490

R. LEMBERG, A. VELINS

Table I I shows that the ferrohaemochromes of monoformyldeuterohaem with different ligands in 2o % ligand-o.o8 N NaOH-Na2S204 were all similar, except that the position of the m a x i m a slightly varied. A ferrohaemochrome-type spectrum was also obtained in 20 % ethanol-o.o8 N NaOH-Na2S204 in the absence of a nitrogenous ligand. In contrast, monoformyldeuterohaem in o.I N NaOH has only one broad a-band at 595-555 mt~ and a y-band at 404 m/x (Table VI); R2 (ea04illll/e~28 nl/,) was below I. The diethylamine and the lutidine compounds were perhaps not fully reduced under these conditions, since it was observed that reduction proceeded slowly. Table I I I shows the absorption spectra in IOO % ligand solution without added TABLE lI MONOFORMYLDEUTEROFERROHAEMOCHROMES

~VITH DIFFERENT

LIGANDS

IN

20°,0

LIGAND

O . O 8 ~x~ ~

NaOH-Na2S204 in m u. i n parentheses e x t i n c t i o n s relative to a - b a n d m a x i m u m .

Haem

Ligand

a-Maximum Minimum

7-Maximum

D i m e t h y l ester D i m e t h y l ester D i m e t h y l ester Free h a e m i n Free h a e m i n Free h a e m i n

~-Picoline fl-Picoline 2,4(5)-Lutidine Ammonia Diethylamine Ethanol

582. 5 578 580 580

428 428 427 433 429 432. 5

551 551 553 55 ° 548 522

577.5 583.5

(0.49-0.60) (0.35) (0.46) (0.43) (o.76) (0.33)

1¢1. R,~**

(5.4 8 5.74) 5.5 (4.92) 7-4 (4 .8I) 3.7 (6.35) 18 (4.o) 1.55 (6.I) 1 i..2

1.28 2.0 1.72 -2-04 1.79 1.64 1.06 2.08

* t{ 1 = ~i'a/e620 In/*. ** R2 ~ ~'e/~40~ Ill/l" TABLE l II ABSORPTION

SPECTRA

OF SOLUTIONS

OF HAEMINS

IN XVATER-FREE

LIGANDS

in mlt. I n parentheses e x t i n c t i o n s relative to a - b a n d m a x i m u m .

Haem

Ligand

a-Maxirnurn Minimum

Haem a Chlorocruoro Monoformyldeutero ester Monoformyldeutero ester Monoformyl deutero ester

Pvridine l@ridine

583.5 573"5

Pyridine fl-Picoline Diethylamine

Monoformyldeutero ester Monoformyldeutero ester Monoformyldeutero ester Monoformyldeutero ester Haem a

)~-3/Iaximum

/~1"

R2**

55 ° (0.28 0.34 ) 429 (5.05) 547.5 (°.4I) 425.5 (5.0 5.82)

8. 4 5.25

1.82 2.5

574

548

(0.30 0.54) 425.5 (6.12)

0.3

1.43- 2.7

573

546

(o.31 o-40) 424.5 (5.93)

7.2

1.39 _,.-,2

573

542.5 (o.60)

426.5 (3.92)

1.85

t.4 ~

409

(8.1-9,o)

1.2o

o.72

41I

(9.9)

1.o3

0.77

407

(IO.2)

1,O

0.75

4 °8

(3-72)

i. 14

o. 75

1.12

o.8o

552 539 Broad 558 2,6-Lutidine Broad 2,4(5)580 555 Lutidine Broad Triethyl575 amine Broad 2,4(5)585 Lutidine Broad

a-Picoline

* R 1 as in Table I. * * ] { 2 ~ 6481 111/~/E414 ln/* f o r h a e m

a;

---

e42s lnlt/~405

Biochim. Biophys. Acta, lO 4 (1965) 487-495

412.5 (3.2)

lilp

tor t h e

two

other h a e m s .

491

FORMYL-SUBSTITUTED HAEMOCHROMES

reducer. It is obvious that these fall into two quite different types. Those in pyridine, fl-picoline, and diethylamine are ferrohaemochrome spectra. They differ from the spectra in dilute ligand reduced by dithionite b y having the a-bands and y-bands at shorter wavelengths (4.5 m/~ for the a-band of haemochrome a, 6.5 m/z for that of monoformyldeuterohaemochrome; 2 m/~ for the ~,-band of haemochrome a, 2.4 m/~ for that of monoformyldeuterohaemochrome); and b y the presence o~ a very weak m a x i m u m at about 525-53 ° m/~. These spectra, like those of Table II, differ entirely from the spectrum of the free haem in alkaline solution (Table VI). In contrast to the ferrohaemochrome spectra in pyridine, fl-picoline and diethylamine, those in a-picoline, the lutidines and triethylamine are clearly not ferrohaemochrome spectra. The spectra show an indistinct a-band and a low R 1 ratio, only slightly above I ; and a y-band at 412.5-414 m # for haem a and 4o7-411 m/z for monoformyldeuterohaem compounds with R 2 below I. The V/a ratio is usually high (9-IO), although on two occasions lower ratios were found; in one instance this was due to turbidity of the solution. They certainly differ from the absorption spectra of the free ferric haematins, less distinctly from the absorption spectra of the free ferrous haems (Table VI). However, the fact that these ligands are able to combine with the haems even in their more dilute solutions rules out the possibility that these spectra are due to uncombined haems.

Oxidation of ferrohaemochromes in aqueous pyridine solutions Other observations also show that the solutions mentioned in the above paragraph are not ferrohaem, but ferrihaemochrome solutions. Like ferrohaemochrome a (ref. i) monoformyldeutero-ferrohaemochrome a is slowly oxidized when aqueous pyridine solutions are allowed to stand at room temperature, or when ferricyanide is added (Tables IV and V) and the resulting spectra resemble those obtained by dissolving the haemins in a-picoline, the lutidines or triethylamine. The somewhat higher values for R 1 in the less dilute solutions (50-80 % pyridine) than in 20 % pyridine (Table IV) are probably due to a partial dissociation of the ferrihaemochrome to free haematin and ligand in 20 % pyridine. Whereas p H has been found markedly to affect the degree of dissociation of ferrihaemochrome a to the free haematinS, 9, no similar effect was found for monoT A B L E IV ABSORPTION SPECTRA OF HAEMINS IN AQUEOUS PY'RIDINE PLUS FERRICYANIDE OR AFTER LONG STANDING IN AIR in m~. I n parentheses e x t i n c t i o n s relative to a - b a n d m a x i m u m . R 1 as in Tables I a n d I l l , R 2 as in Table I I I .

Haem

Ligand

a-Maximum y-Maximum R 1

5 0 - 8 0 % pyridine + ferricyanide 587 or after long s t a n d i n g Haem a 20-3o % pyridine after long 587 standing Monoformyldeutero 2 o % p y r i d i n e - o . o 8 N N a O H 580 ester Chlorocruoro ester 2 0 % p y r i d i n e - o . o 8 N N a O H 58o Chlorocruoro ester 5 o % p y r i d i n e + ferricyanide 579 Haem a

R2

414

(8.1)

1.62

o.88

414

(7.2)

i.o8

o.78

412.5 (5.1)

1.47

o.88

412.5 (5.4) 416 (7.7)

1.17 1-77

o.79 o.96

Biochim. Biophys. Acta, lO4 (1965) 487-495

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R. LEMBERG, A. VELINS

TABLE V A B S O R P T I O N S P E C T R A O F H A E M I N S I N A Q U I ~ O U S P I C O L I N N S , A M M O N I A OR A L C O H O L

). in m/,. In parentheses extinctions relative to wavelength indicated. R 1 as in Tables I and Ill, t ~ as in Table [II. Haem

Ligand

3laximum in visible

7 Maximum

171

1¢2

Monoformyldeutero ester

20% :¢-picoline-o.i N NaOH

~ 574 (i.o)

400 (8.5)

J,o6

0.7o

Monoformyldeuteroester

2o°0 c¢-picoline water

~ 574 (i.o)

404.5 (6.0}

i.o 3

o.71

Monoformyldeutero ester

80 % a-picoline-water

~ 574 (i.o)

404

(8.0)

~.~8

o.70

Monoformyldeutero ester

20% fl-picoline-water

020 (I.O)

400

(7.8)

t.o

0.09

Monoformyldeutero

16 % NHa--o.o 5 N NaOH

583 (I.O)

400

(7.8)

r. t7

o.75

(free acid)

20°0 a l c o h o l - o . o 8 N N a O H

584 (i.o)

402

(8.8)

i.o 5

0.74

f o r m y l d e u t e r o h a e m o c h r o m e e s t e r (Table V), p r o b a b l y b e c a u s e t h i s was in t h e e s t e r form. A n o t h e r difference b e t w e e n h a e m a a n d t h e o t h e r f o r m y l h a e m s has b e e n t h a t , in c o n t r a s t to h a e m o c h r o m e a (refs. x, 2) no r e d u c t i o n to f e r r o h a e m o c h r o m e is f o u n d on e v a c u a t i n g a s a m p l e of p a r t l y o x i d i z e d m o n o f o r m y l d e u t e r o h a e m o c h r o m e in 5 o - 8 o % p y r i d i n e . T h e e x p e r i m e n t s h o w n in T a b l e V I I i n d i c a t e s t h a t t h e r e d u c t i o n of ferrih a e m o c h r o m e a is d u e to t h e p r e s e n c e of r e d u c i n g i m p u r i t i e s in h a e m i n a. W h e n small a m o u n t s of a d i l u t e f e r r i c y a n i d e s o l u t i o n w e r e r e p e a t e d l y a d d e d t o a s o l u t i o n of h a e m i n a in i o o % p y r i d i n e , t h e e x t i n c t i o n s at 43o m/z (7) a n d 6o5 rot* (~) i n i t i a l l y d e c r e a s e d a f t e r e a c h a d d i t i o n of t h e f e r r i c y a n i d e solution, b u t rose a g a i n on s t a n d i n g . S i m i l a r l y t h e r a t i o e430 m,/e4~ ~ ~ , fell i n i t i a l l y b u t i n c r e a s e d a g a i n on s t a n d i n g a l t h o u g h it was n o t f u l l y r e s t o r e d to its o r i g i n a l v a l u e . O n l y a f t e r a b o u t 1. 4 m l of t h e f e r r i c y a n i d e s o l u t i o n h a d b e e n a d d e d to 3 m l of t h e p y r i d i n e , d i l u t i n g it to a b o u t 68 °,'o, c o m p l e t e o x i d a t i o n o c c u r r e d . E v e n t h e n , p a r t i a l r e d u c t i o n c o u l d be o b s e r v e d a f t e r k e e p i n g t h e s o l u t i o n for 16 h in an e v a c u a t e d T h u n b e r g t u b e t h e s t o p p e r of w h i c h c o n t a i n e d s o d i u m d i t h i o n i t e . I t a p p e a r s u n l i k e l y t h a t this e x p e r i m e n t can be e x p l a i n e d as oxygenation followed by deoxygenation. TABLE V1 ABSORPTION

S P E C T R A OF H A E M A T I N S A N D H A E M S

IN o.~ N NaOH

). in m u. In parentheses extinctions relative to wavelength indicated. Haem

Valency

Haem a

Ferric Ferrous Ferric Ferrous

Monoformyldeuterohaem

635 (i.o) 59o--6oo (1.o) 62o (i.o)*** 595-555 (I.o)

R,, * *

o.68-o,91 1.48-1.69 1.o4 1.5o

o.t)4-o.75 0.85-0.97 0.57 0.87

• R1 = e~88 m~/e6a0 mt~ for haem a; e~sI m~/e620m~ for monoformyldeuterohaem. e 4 3 1 i11/~/~414nlH for haem a; e42s.5 m~de4o5 mu for monoformyldeuterohaem. * No distinct maximum.

• * R 2 = • *

40o 405 (6.2-7.1) 4o8-41o (6.0-8.7) 39z (11.6) 404 (lo.4)

R 1*

Biochim. Biophys. Acta,

lO4 (1965) 487-495

FORMYL-SUBSTITUTED

493

HAEMOCHROMES

TABLE Vll EFFECTS OF REPEATED ADDITIONS OF SMALL AMOUNTS OF DILUTE SOLUTIONS OF POTASSIUM F E R R I C Y A N I D E ON T H E A B S O R P T I O N S P E C T R U M O F A S O L U T I O N O F H A E M I N a I N P Y R I D I N E

Total ml

Pyridine

ferricyanide solution added

after addition (%)

o. I ml

ioo 97 97 94 94 91 91 88 88 83.5 83.5 79 75 71.5 71.5 71.5 68 68

0.2 ml 0.3 ml 0. 4 ml 0.6 ml 0.8 m] i.o ml 1.2 ml 1.4 ml

8430 m/~

~430 I1~/~414 m/* 8605 rn/~

Remarks

I.OI 0.95 I.OO o.973 0.990 o.988 o.993 o.97 0.975 0.935 o.94 0.908 0.883 0.86 o.87 0.92 o.61 O.TO

1.44 1.3o 1.37 1.33 1.37 1.365 1.35 1.31 1.32 1.28 -1.16 I.IO 1.o7 1.o75 1.21 0.825 1.o3

Before addition Immediately after addition After 26 rain Immediately after addition After 13 rain Immediately after addition After 17 rain Immediately after addition After 20 rain Immediately after addition After 2o min

0.22 o.21 o.223 o.21 0.23 o.198 o.213 --o.198 -o.187 o.17 o.163 o.165 o.178 o.135 o.16

Immediately after addition After 12 rain After 16 h evacuation Immediately after addition After 16 h evacuation

DISCUSSION

I t has been shown t h a t in contrast to protohaemin, h a e m i n s with a formyl side chain are dissolved in p y l i d i n e to form solutions of ferrohaemochromes. The same holds for solutions in ~-picoline a n d in diethylamine, b u t n o t for other nitrogenous ligands such as a-picoline, lutidine or triethylamine. The electron-withdrawing formyl group is k n o w n to increase the o x i d a t i o n - r e d u c t i o n potential of haem compounds 1°-1~. This is p r o b a b l y the cause of the different b e h a v i o u r of p r o t o h a e m i n from the formylhaemins. I t is, however, n o t yet clear whether the electron donor in this reaction is the ligand itself or impurities present in it. These need n o t be present in a m o u n t s m u c h larger t h a n the small a m o u n t of haemin. If pyridine itself is the electron donor, its oxidation p r o d u c t m i g h t have been x,e'-dipyridyl. This appeared to be supported b y the observation t h a t on addition of water a n d dithionite there was sometimes a gradual increase of the absorption in the green part of the spectrum, which m i g h t have been due to slow c o m b i n a t i o n of e,a'-dipyridyl with traces of ionic iron present as cont a m i n a t i o n . The differential spectrum was, however, n o t t h a t of ferrous ~,~'-dipyridyl b u t i n d i c a t e d the reaction of the formyl side chain with sulphur dioxide 1. On dilution with o.I N N a O H i n s t e a d of with water, no increase of the absorption in the green p a r t of the spectrum was found, although the dipyridyl iron reaction was n o t p r e v e n t ed. Moreover, such a n e x p l a n a t i o n does not appear likely for the reaction of monof o r m y l h a e m i n with diethylamine. While distillation of the pyridine over p e r m a n g a n a t e failed to p r e v e n t the reduction to monoformyldeuteroferrohaemochrome, we have in one instance observed only partial reduction b y dissolving the h a e m i n in such a pyridine after it had stood exposed to air for several weeks after the distillation over p e r m a n g a n a t e . T h e reduction of the ferric iron of h a e m i n is therefore more likely caused Biochim. Biophys. Acta, lO4 (1965) 487-495

494

R. LEMBERG, A. VELINS

by impurities in the pyridine. The reduction of haemin a in pyridine is undoubtedly due to a large extent to impurities present in the haemin a itself rather than in the pyridine, as the experiment reported in Table VII and the difference between the effect of evacuation on partly oxidized haemochrome a and monoformyl deuterohaemochrome show. The reduction of partially oxidized ferrohaemochrome a in aqueous pyridine by evacuation therefore does not prove the formation of a reversible oxygen compound of pyridine ferrohaemochrome a. Reaction of such a compound with ferricyanide could only result in its irreversible oxidation to a ferrihaemochrome, yet evidence for reduction by evacuation has been found even after oxidation by ferricyanide. The difference of the nature of the ligand on the spectra of the haemin solutions in these ligands clearly indicates the importance of a steric factor. Of particular interest is the great difference found between fl-picoline on the one hand and a-picoline and the a-substituted lutidines on the other. It appears that one methyl substituting an a-position of pyridine suffices to prevent the ferrohaemochrome formation. Again the difference between diethylamine and triethylamine may be due to steric hindrance. It would be easier to understand this steric hindrance if the non-ferrohaemochrome spectra found in a-picoline, the lutidines and triethylamine were the spectra ot the free haems, but for various reasons enumerated above, it appears that these spectra are spectra of ferrihaemochromes, possibly with some admixed free haematin, but not ferrous haem spectra. The steric factors may, however, influence the oxidationreduction potential by differential affinity of the ligands to ferrous and ferric haem, and thus the degree of reduction of the ferric haemin dissolved in the ligand. Table V does not indicate great differences in the affinity of ferric haematin to the ligands, with the exception of 4-methylimidazole. It therefore appears likely that the predominant steric effect is on the affinity of ferrous haem to the ligands. Indeed, the pK of dissociation of a-picoline protoferrohaemochrome has been found to be considerably lower (pK 4) than that of the corresponding pyridine haemochrome, 6.3 (see ref. 13). TABLE VIII pKa oF LIGANDBASE~ pKa's were determined at z5° (see ref. i6, 17). Base

pKa

Pyridine fl-Picoline ~-Picoline 2,4(5)-Lutidine 2,6-Lutidine Ammonia Triethylamine Diethylamine

5.17 5.68 5-97 6.5 I-6.79 6.75 9.25 1o. 67 lO.99

Differences in the basicity of the ligands (Table VIII) which according to FALK AND PERRIN14 and FALK15 (cf. ref. 16) influence the oxidation-reduction potential, cannot explain the results. While it is true that the pKa's of pyridine and fl-picoline are Biochim. Biophys. Acta, lO4 (1965) 487-495

FORMYL-SUBSTITUTED

495

HAEMOCHROMES

slightly lower than those of u-picoline and the two luticlines, the pKa of cliethylamine much higher and even higher than that of triethylamine.

is

NOTE

ADDED

IN PROOF

On a re-investigation of the absorption spectrum of monoformyldeuterohaem in alcoholic NaOH, we have found that this is not a haemochrome spectrum as reported in the last line of Table II, but is, in fact, rather similar to the spectrum of monoformylcleuterohaem in 0.1 N NaOH, reported in the last line of Table VI : a-band 600 rnp (I.o), y-band 404 rnp (5.7), R, 1.25, R, 1.0. Similar small differences, perhaps due to change in polymerisation, have been found between the absorption spectra of protohaem in aqueous and alcoholic alkali. Received April moth, 1965 ACKNOWLEDGEMENTS

This work has been supported by a Public Health Research Grant No. HEO7827 from the National Heart Institute of the United States of America and by the National Health and Medical Research Council of Australia.

REFERENCES R. LEMBERG, Biochem. Z., 338 (1963) 97. W. J. CAUGHEY AND J. L. YORK, J. Biol. Chem., 237 (1962) PC2414. D. B. MORELL, J. BARRETT AND P. S. CUTZY, Biochem. J., 78 (1961) 793. M. J. PARKER, Biochim. Biophys. Acta, 35 (1959) 496. R. LEMBERG AND J. PARKER, Australian J. Exptl. Biol. Med. Sci., 30 (1952) 63. R. LEMBERG, D. B. MORELL, N. NEWTON AND J. E. O’HAGAN, PVOC. Rqy. Sot. London, Ser. B. I55 (1961) 339. R. LEMBERG AND J. E. FALK, Biochem. J., 4g (1951) 674. P. S. CLEZY AND D. B. MORELL. Biochim. Biobhvs. Acta. 71 f1961\ 165. R. LEMBERG, in F. F. NORD, Advan. Enqmol.: 2; (rg61).;65.‘ - -’ R. W. HENDERSON, Ph.D. Thesis, University of Melbourne, 1960. J. E. FALK, Porphyrins and Metallo$or$hyrins, Elsevier, Amsterdam, 1964. R. LEMBERG AND T. W. LEGGE, Hematin Cornbounds and Bile Picments. Interscience, New

York, ww. p.

w.-

‘3 H. F. HOLDEN AND M. FREEMAN, Australian J. Exptl. Biol. Med. Sci., 6 (1929) 79. I4 J. E. FALK AND D. D. PERRIN, in J. E. FALK, R. LEMBERG AND R. K. MORTON, Haematin Enzymes, Pergamon, London, 1961, p. 70. and Metallopovphyrins, Elsevier, Amsterdam, 1964, p. 70. 15 J. E. FALK. Porphyrins Vol. g, Elsevier, 16 J. N. PHILLIPS, in M. FLORKIN AND E. H. STOTZ, Com$rehensive Biochemistry, Amsterdam, 1963, p. 62. 17 C. LONG, Biochemists’ Handbook, 1961, Table 6. Biochim.

Biophys.

Acta,

104 (1965) 487-495