- Email: [email protected]
Anomalous rotary dispersion of metachromatic mucopoly-saccharide-dye complexes II. Heparin-Methylene blue complexes at acidic pH
56
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 737 ANOMALOUS ROTATORY D I S P E R S I O N OF METACHROMATIC MUCOPOLYS A C C H A R I D E - D Y E COMPLEXES II. H E P A R I N - M E T H Y L E N E B L U E COMPLEXES AT ACIDIC p H AUI)I~EY L. STONE AND H E R B E R T MOSS* Laboratory of Neurochemislry, National Institule of )3enlal Health, Bethesda, Md. 2ooz 4 (U.S.A.) (Received J u l y I8th, I956)
SUMMARY
The phenomenon of induced optical rotatory dispersion (induced Cotton effect) in symmetric planar dyes when bound to certain asymmetric polyanions has been further investigated for methylene blue-heparin complexes in order to determine the source of the a s y m m e t r y in the bound dye aggregate. Induced rotation was measured as a function of p H in the range of ionization of the carboxyl group of heparin. Spectrophotometric titrations showed that at p H 2.4 essentially all the carboxyl groups were unionized while at p H 3.1 about 2o % of the carboxyl groups were negatively charged. The metachromasy of the stoichiometric complexes changed towards the orthochromatic color upon loss of the carboxyl-group binding site. Analysis of the metachromatic spectra according to quantitative parameters relating to dye-dye interactions showed, however, that although hypochromism (loss of oscillator strength) was decreased at p H 3.1 and 2.4 there was no concomitant decrease in the metachromatic shift in center of gravity. The rotatory dispersion curves were found to increase in magnitude with decreasing p H rather than to correlate solely with the decrease in dispersion interactions (hypochromism) or with the similarity of the degree of exciton-type dye-dye interactions (frequency shifts), showing the complex nature of the induced Cotton effect at p H 6. 7. The increase in magnitude of the effect from p H 6. 7 to 2.4 can be explained by the loss of a negative, dispersion-type Cotton effect which cancels negative and positive rotations of an exciton-type effect at p H 6. 7, or it could be the result of a conformational change in the asymmetric polymer-dye aggregate. The correlation of the source of the induced rotation at least in part with dye-dye rather than dye-polymer and/or dye-solvent interactions further supports the interpretation that the anionic sites of heparin in solution are in helical order. A molecular model of heparin is presented showing the helical order possible for c¢-(i-~ 4)-glucose polymers. The specific conformation is stabilized by a linear hydrogen bond involving the nitrogen of the sulfamino group as the acceptor and the C-3 hydroxyl group of the following uronic acid as the donor. Certain features of this molecular conformation are discussed in relation to the biological activity of heparin. * Visiting Chemist from High School Science Teacher Study-Work Institute, Summer, 1964
Biochim. Biophys. Acta, 136 (1967) 56-66
COTTON EFFECTS IN HEPARIN--METHYLENE BLUE COMPLEXES
57
INTRODUCTION
For over a decade secondary features of heparin structure have been suggested and alluded to in relation to its manifold biological activities even though knowledge of its primary structure was skeletal at the first proposals 1-6. With the rapid advance in understanding of the primary structure during the last few yearsT, s, such proposals still hold and m a y be approached in a more specific fashion. Two separate aspects of secondary structure possible for heparin are (i) helical order due to the ~ - ( I - ~ 4 ) D-Glc repeat configuration, analogous to the amylose structuree, 8 and (2) a linkage between the sulfamino function and an unsubstituted - C - O H , e.g., the "intramolecular sulfate bridges - N - S O 2 0 C - " (refs. I, 4, 5). In regard to the first feature, helical order for the anionic sites of heparin in solution was indicated b y the demonstration 9 that four, chemically different, planar symmetric dyes exhibit induced anomalous optical rotatory dispersion in the metachromatic absorption band when bound to this polyanion. It was shown that the induced Cotton effect was dependent upon the metachromatic reaction of the dyes and upon the integrity of the heparin molecule, particularly in regard to the sulfamino group. The data showed that the dyes must be bound, but in addition, that they must be bound to nearby sites, supporting the view that the induced a s y m m e t r y was largely dependent upon dye-dye rather than dye-polymer interactions. Induced Cotton effects have been predicted for chromophores in a helical array using various theoretical models 1° ,6. The exciton-coupling model '2,'3 predicts a non-symmetric rotatory dispersion curve with a dominant feature, the peak or trough, flanked by two minor troughs or peaks, respectively, giving a net change in rotation equal to zero. The coupling of different electronic bands among chromophores (dispersion interactions) results in a symmetric Cotton effect in the visible or ultraviolet absorption region which is compensated by an equal and opposite Cotton effect in a shorter-wavelength absorption band so that an apparent net rotation m a y occur in any one absorption band '2. The non-symmetric shape of the curve for the methylene blue-heparin complexes also supported the hypothesis that the induced rotation derives largely from dye-dye rather than dye-polymer interactions. Further, it was found that the addition of histamine to the complex caused a small increase in induced rotation. Hence the ordered array of dyes leading to the induced rotation was apparently not destroyed b y the addition of histamine to a site on the heparin molecule. Insofar as the role of the sulfamino group in the secondary structure of heparin is concerned, hydrolysis of about 33 % of the N-sulfates which is known 4, ~ to cause a loss of almost all the anticoagulant activity, led to a marked loss of the induced a s y m m e t r y in bound methylene blue with relatively slight loss of metachromasy. The data might be interpreted as indication of a change from a "native" molecular conformation if the source of the induced rotation were known in regard both to the pertinent binding sites and to the dye-dye interactions, since exciton-type interactions between neighboring dyes would be directly dependent upon intersite conformations. In the present study the role of the carboxyl group in the induced rotation of the methylene blue-heparin complex was investigated. The results showed that changes in metachromasy and induced rotation of the dye-aggregate (or polymer-site geometry) occur upon ionization of the carboxyl group which support the interpretaBiochim. Biophys. Acta, 136 (I967) 5 6 - 6 6
58
A . L . STONE, H. MOSS
tion that at least part of the induced rotation at neutral pH is due to dye-dye interaction and hence to a helical array of anionic sites. Furthermore, a molecular model of heparin was constructed, using Courtauld atomic models, which shows secondary structural features consistent with the induced anomalous rotation of the dye complexes. MATERIALS AND METHODS
The spectrophotometric techniques involving polymer-dye complexes were described previously 17-21. Samples of heparin and methylene blue were the same preparations as in the previous studies 9,17. Histamine was obtained as the dihydrochloride from Nutritional Biochemicals, Inc. The concentration of the dye and heparin anionic sites was about I. 4. lO.5 M at all pH values with absorbance less than I.O unless otherwise stated. Buffers were I mM potassium phthalate at pH 2.4 and 3.1 and I mM sodium cacodylate at pH 6. 7. Measurements of rotation were made on a Rudolph spectropolarimeter model 2oo using a symmetrical angle of 1.8, pathlength of 2.5 cm and fused quartz cells made by Opticett Co., Inc. Results with the Rudolph spectropolarimeter have yielded anomalous rotation dispersion curves with sharper troughs and smaller amplitudes than the Bendix spectropolarimeter 9, which was thought to be due to a narrower band pass in the former instrument. The optical parameters of the metachromatic reaction relative to those of the free dye were obtained as described previously ~2 from calculation of the oscillator strength (f), the center of gravity and the band half-width of the various spectra using program HJDoo8C (written by Dr. H. DEVOE) in the Honeywell 8oo computer. Molecular models were made with the Courtauld atomic models (Ealing Corp., Cambridge, Mass.) with the relative scale of o.8 incer per A. RESULTS
Induced anomalous rotatory dispersion curves for stoichiometric methylene blue-heparin complexes at pH 6. 7, 3.1 and 2.4 are shown in Fig. I. Stoichiometry based upon titration end points 17 indicated that about 80 % of the carboxyl groups were unionized at pH 3.1 and IOO % at pH 2. 4 under these conditions. It can be seen that relative to pH 6. 7 the induced rotation of the dye is not reduced when the carboxyl site is removed, but is increased and slightly displaced towards a higher wavelength. It is noteworthy that the three curves intersect at a common "isorotation" point, 566 m/x. Although the curves do not represent the carboxyl ionization per se, they represent a change in the structure of the methylene blue-heparin complex due to this ionization. A series of curves with a common non-zero point of rotation has been reported recently for the double-helix to single-chain-helix transition of a polynucleotide ~ and for the "stacked" to "unstacked" transition in single-strand trinucleotides ~4. Table I summarizes the values of the various optical parameters (see text in table). The oscillator strength is a measure of the total intensity of the absorption band while the center of gravity and the band half-width give the intensity-weighted average frequency and the frequency distribution of the band. Changes in these parameters as compared with those for the free dye are quantitative measures of the Biochim. Biophys. Acta, 136 (1967) 56-66
COTTON +10
EFFECTS
IN
HEPARIN--METHYLENE
I t I I I I I I I I I I I I I
BLUE
COMPLEXES
59
I J ~ I I I i r I I I
3 + 5--
/
~ 3.-w". . . .
~
~----t 2
- 5--
g ,%
~ -I0--15 --
-20 --
I _25 I I I I I ] I I I r I I f t P I 450 500 550 600 m~
I F I I I I I I I I I 650 700
I 750
F i g . I. I n d u c e d o p t i c a l r o t a r y d i s p e r s i o n o f m e t h y l e n e blue-heparin with ionization of carboxyl g r o u p s , q~ ~ m o l a r r o t a t i o n o f t h e d y e - - I o n / M / w h e r e M is t h e m o l a r i t y o f t h e d y e s o l u t i o n , the observed rotation and 1 the pathlength i n d e c i m e t e r s . C u r v e i a t p H 6 . 7 ; C u r v e 2 a t p H 3.1 ; C u r v e 3 a t p H 2. 4. T h e b r o k e n l i n e s i n d i c a t e i r r e g u l a r i t y in e x p e r i m e n t a l points.
degree of metachromasy 22, Acg (frequency shift) giving an estimate of the strength of the interaction between like transition moments of the bound dyes and Af (hypochromism) giving the extent of interaction between non-identical transition moments of the dyes, and solvent or dye and polymer. The ratio of absorption at 66o m/~ to that at 61o m~, the ~//3 ratio, which has often been used as a measure of metachromasy, is also included. However, recent evidence 22 shows that for most polymerdye systems this ratio does not parallel 2cg (or/If) in magnitude and therefore cannot be taken as a comparative measure of metachromasy from a theoretical viewpoint. The values of the spectral parameters of the free methylene blue (Table I) do not change with pH. It might be expected that metachromasy will decrease with a decrease in anionic site density and it can be seen t h a t / I f and A~/~ decrease as the carboxyl group site is lost, going toward the bluer, or orthochromatic color for the solution of the complex. However, A cg and ABW do not decrease. The former increases slightly at p H 3.1 while the latter is increased at p H 2.4 and 3.1. Thus, "decreased metachromasy" in this case consists of a loss of hypochromism without concomitant decrease in frequency shift. Addition of histamine to the complexes at p H 2.4 at ratios H i / H of 1. 7 or 5 caused a small decrease in all the metachromatic parameters. At a ratio of 22, however, greater decreases in metachromasy occurred in all the optical parameters with a relatively slight decrease in Oa. Comparison of the various parameters at the three p H values are made in the last 6 columns of Table I based upon the values of the p H 6.7 complex as IOO %. Biochim. Biophys. Acla, 1 3 6 ( I 9 6 7 ) 5 6 - 6 6
I
Do
h~
PARAMETERS
OPTICAL
OF METHYLENE
BLUE
AND
METHYLENE
BLUE
tIBPARIN
COMPLEXES
pH
6. 7 3-1 2. 4
6. 7 3.1 2.4
2. 4 2. 4 2. 4
Sample
MB MB MB
MB-H 3IB H MB-H
MB-H-Hi MB-H-Hi MB-H-Hi
1. 7 5.0 22.0
-
-
[5-1
[Hi]
o.133 o.13o O.ll 3
o.228 o.179 o. I5O o.81 o.76 o.51
o.84 0.89 0.83 °-42 o.41 0.30
o.26 0.38 0.39
A B IV
Acg
t .3 8
(£~/~Z 1 )f
BW
<'If
3)
1.33 i "35
i X IO
16.o6 15.99 15.97
(£m
cg
0.665 o.67o o.675
f I0
1.92 1.9o 1.88
c~/fl
o-35 o.32 o.18
o.74 0..56 o.42
A~I~
"3)
278 28o 253
i86 237 282
585 585 585
581 583 585
~ At~ (× zo ~) (mu)
5)5 5t5 545
(my)
2,p
1oo 74 66
58 57 5°
~oo 79 66
.4~ionic .l/ sites
47 43 22
Ioo 76 56
I~I~
61
97 90
IOO lo6 99
"Icg
161 155 i15
IOO 146 15o
149 151 ~36
IOO 12"~ 152
I B IV ~Pa
Per ce~t of resultams qf parameters (fill 6.7)
T h e o p t i c a l p a r a m e t e r s h e r e a r e for m e t h y l e n e b l u e - h e p a r i n c o m p l e x e s c l o s e t o P / D -- i w h e r e P / D is t h e r a t i o of a n i o n i c s i t e s t o d y e m o l e c u l e s . M e t a c h r o m a s y (./If, Acg, A B W ) in t h e m e t h y l e n e b l u e h e p a r i n s y s t e m is g r e a t e r a t h i g h e r r a t i o s (1>/19 = 2 4) a t t h e t h r e e p H v a l u e s . (.:.oncentratiol: of d y e s a n d a n i o n i c s i t e s for a l l t h e c o m p l e x e s is a b o u t 1. 4. 1o -5 M. O p t i c a l p a r a m e t e r s as p r e v i o u s l y r e p o r t e d j7 a r e s y m b o l i z e d a s f o l l o w s : f, o s c i l l a t o r s t r e n g t h ; cg, c e n t e r of g r a v i t y ; B\V, b a n d h a l f - w i d t h in I o -a r e c i p r o c a l c e n t i m e t e r s ; ~//~, t h e r a t i o of a b s o r b a n c e a t t h e ~ b a n d (665 lnjt) t o a t 3 s o r b a n c e a t t h e fl b a n d (61o m u) of t h e a b s o r p t i o n s p e c t r u m ; .If, .)cg, A B W ,
l
TABLE
©
;:u
bl
o
>
o
62
A . L . STONE, H. MOSS
Fig. 2. Views of a m o d e l of h e p a r i n c o n t a i n i n g two dimeric r e p e a t i n g units. (a) O u t s i d e s u r t a c e s h o w i n g spiralling p y r a n o s e o x y g e n s (p) a n d t h e c a r b o x y l a n d t h e (C-3)-O sulfate groups. T h e C-6 h y d r o x y l g r o u p of t h e g l u c o s a m i n e is p o i n t i n g t o w a r d s t h e reader. T h e O-sulfate g r o u p could be placed on t h e C-6 h y d r o x y l g r o u p i n s t e a d of t h e C-3. I n t h e model, t h e C-6 h y d r o x y l is clearly available for e t h e r linkage to protein. (b) Inside surface s h o w i n g t h e groove, spiralling glycosidic o x y g e n s (white dots), linear h y d r o g e n b o n d b e t w e e n t h e a m i n o s u g a r n i t r o g e n (acceptor) a n d t h e C-3 h y d r o x y l of t h e following uronie acid. T h e d i s t a n c e available in t h e groove b e t w e e n t h e Nsulfate a n d p r e c e d i n g c a r b o x y l g r o u p is a b o u t 7.5-8 ~k w h i c h can r e a d i l y a c c o m m o d a t e h i s t a m i n e with t h e far ring n i t r o g e n n e a r t h e c a r b o x y l a n d t h e a m i n e n i t r o g e n n e a r t h e sulfate.
Biochim. Biophys. Acta, I36 (1967) 5 6 - 6 6
COTTON EFFECTS IN HEPARIN--METHYLENE BLUE COMPLEXES
63
Molecular models Fig. 2 shows several views of a model of heparin based upon an a-(I --~ 4)glycosidic repeating linkage with the normal chair conformation in the sugar rings 25 and the 0-sulfate on C-3 of the glucosamine moiety. Placing the O-sulfate on C-6 tends to put this sulfate group more on the outside of the molecular contour (see Fig. 2a where the C H 2 0 H points toward the reader) but does not alter fundamental relations between the repeating units. As was shown previously by VELLUZ6 with molecular models of hexamesitylene-heparin complex, the heparin molecule can assume a helical or spiral form anticipated from the amylose-like repeating unitsT, s. There is some flexibility to the molecule, but a specific conformation would be stabilized b y the formation of a hydrogen bond between the C-3 hydroxyl of the glucuronic acid moiety and the nitrogen of the preceding glucosamine ring. This bond is readily formed in the model and appears to be the only linear hydrogen bond possible for the spiral conformation. Such - O H . . . N bonds have been described in the crystalline structures of several compounds, e.g., p-aminopheno126 and hydroxylamine 27, and appear to be reasonably strong, of the order of magnitude of hydrogen bonds which occur in amides and peptides 28. The views of the model (which consists of two dimeric units) show both the twisting of the glycosidic oxygens close to the axis of the molecule (dots) (Fig. 2b) and the spiralling of the pyranose oxygens (P's) along the helix (Fig. 2a). It can be seen that the negative binding sites for the cationic dyes (0-sulfate, N-sulfate and carboxyl) would impart a helical order to a bound dye aggregate, which could give rise to the observed induced Cotton effect(s). Furthelanore, one can readily place a model of the histamine molecule in the groove caused by the spiralling of the molecular surface. In this position the histamine can span the groove between the N-sulfate and the preceding carboxyl sites with its primary amine end at the N-sulfate and its ring nitrogen end near the carboxyl group. Furthermore, there are two axial hydrogens in the groove which constitute a small area of non-polarity which could interact with the imidazole ring. A model of serotonin could be seen to fit less readily than histamine in the heparin groove, since it is more bulky and over 2 A longer.
DISCUSSION
The phenomenon of induced anomalous rotation in methylene blue-heparin complexes could be interpreted relative to polymer conformation if it could be shown that the induced Cotton effect is due to dye-dye interactions among bound cationic dyes, which represents the spacial orientation of fixed substituents on the polymer chain. Previously 9 it was shown that: (I) the phenomenon is generally observed among chemically different cationic dyes bound to heparin, rather than peculiar to methylene blue; (2) it is a property of bound dyes rather than of the free dye in a solution of an optically active polymer since competitive inhibition of ~binding by bivalent ions diminished asymmetric metachromatic interactions; and finally (3) d y e dye interactions are involved since distribution of bound dyes among m a n y excess sites causes concomitant shifting and ultimate disappearance of the a s y m m e t r y and metachromasy which would not be expected for dye-polymer interactions alone. The present study shows that the induced rotation is due substantially to exciton-type Biochim. Biophys. Acta, 136 .(1967) 56-66
64
A. L. STONE, H. MOSS
dye-dye interaction which permits further interpretation of the induced rotation and conformational aspects of the polymer. In principle the induced rotations exhibited by dye-polymer complexes can be analyzed for their exciton, dispersion, and other possible components in a manner analogous with the separation of hypochromism and frequency shift. Such quantitative calculations are not made in this report because the magnitude of the error in rotations above 62o m/~ preclude the evaluation of an integrated value for rotation, i.e., rotatory strength. Measurements are in progress using a photocell more sensitive at the upper wavelengths. Qualitatively, however, irregularities in induced rotation curves near the trough or peak regions which have been observed in this and other systems18,19, 29 indicate the complex nature of the Cotton effects.
Hypochromism The Af (Table I) decreases by one-third as one out of the three sites available per dimeric sugar unit at p H 6. 7 is no longer occupied b y dye, showing that dispersiontype interaction occurs between dyes bound to the carboxyl sites and other dyes, the polymer and/or the solvent. Furthermore, the percentage in Af follows closely the percentage change in total anionic sites. The Cotton effect, however, is not reduced, showing lack of direct correlation between Af and the induced rotation and indicating further that the effect is the resultant of more than one type of interaction. If the dispersion interaction at p H 6. 7 gave rise to a negative Cotton effect in the visible band, its positive contribution would cause negative rotations on the lower-wavelength side of the resultant curve to be less than at p H 2,4, which is what is observed. The possible presence of a small number of L-iduronic acid residues in the primary structure of heparin 3° m a y prove to have bearing on the magnitudes of the resultant effects at the various p H values if dye-polylner interactions are important in the hypochromic effect.
The frequency shift It has been found12,13 that generally as the average number of chromophore units in an exciton interaction increases, both the frequency shift and the Cotton effect increase in magnitude, all other factors being equal. In this system, the 2cg might be expected to decrease from p H 6.7 to p H 2. 4 as the repeating number of bound dyes is reduced from 3 to 2 if all other factors remained the same. However, the Acg and the induced Cotton effect are not diminished while there is a decrease in Af supporting the argument that a substantial component of the Cotton effect is exciton induced and that other factors in addition to the average distance between the bound dyes are involved. Since the angular positions of the dyes in the aggregates (geometric factors) are also paramount in the net electronic interactions, the data suggest that the local neighboring dye-polymer conformations differ in the acid and neutral complexes. The change in the asymmetric metachromasy with p H might, therefore represent a conformational change in heparin rather than solely a change in the relative proportions of the various component parts of the resultant Cotton effect. L-Iduronic acid moieties 3° would be expected to affect the average geometric factors at p H 6. 7 and contribute to the observed changes with pH. However, such moieties, if present, would constitute a minor component of the primary structure. Biochim. Biophys. Acta, 136 (1967) 56-66
COTTON EFFECTS IN HEPARIN-METHYLENE BLUE COMPLEXES
65
Recent studies with amylose and carboxymethylamylose31,32indicate that these ~-(I~4)-glucose polymers undergo conformational changes in solution with change in pH. Carboxymethylamylose undergoes changes upon ionization of the carboxy! group (pH 2-6) which is believed to represent a helix-to-coil transition 32, however, amylose is believed to have helical order in both acid and neutral pH. The fact that there is an induced Cotton effect for methylene blue-heparin at pH 6. 7 indicates that some helical order exists among the sites in the heparin molecule at that pH as well as at pH 2.4. If at neutral pH the helical nature is partial or segmented as proposed for amylose 31, then the transition corresponding to that in CM-cellulose might be to a more rigid type of helix. The results of the addition of histamine to methylene blue-heparin complexes at pH 2.4 indicate that at ratios of Hi/H of about 2 and 5 little dye is displaced from sites available in the absence of histamine since only slight effects were seen in metachromasy and induced rotation. Studies on the extent of binding of histamine in this system would be necessary before any further conclusions could be reached. Molecular model In the past, secondary structural features have been ascribed to the N-sulfate group in terms of a "sulfate bridge"l,4, 5, -N-SO2-O-C-. The available chemical data, however, showed that all the sulfates were titratable 4,5. The present model (Fig. 2) proposes that the N-sulfate grouping is involved in the secondary structure of the molecule through hydrogen bonds between the amino nitrogen (aeceptor) and the C-3 hydroxyl of the following uronic acid. Molecular models show this structure to be readily formed and such O - H . . . N bonds have been described and can be reasonably strong 2s. Recent crystallographic analysis of the cyclohexaamylosepotassium acetate complex 33, which would be analogous to one turn of an amylose helix, shows a hydrogen bond between the (C-2)-0 and (C'-3)-O of the next sugar ring. This corresponds with the (C-2)-N... H-O-(C'-3) bond proposed above. Molecular models of polysaccharides show small areas of non-polarity which result from the twisting of the sugar rings relative to one another. In the case of fi-linked polymers such as the chondroitin sulfates A and C (where hydrogen bonds can also be formed, i.e., (C-3)-O-H... N-(C'-2) this region includes four C-H groups. The concept of non-polar regions in polysaccharide structure was brought out by FREUNDENBERG~4 (and discussed by BEAR35) from models of amylose. He considered the amylose molecule to be a helix with a center essentially "hydrocarbon" in nature accounting for the adsorption of 12 in the amylose-I 2 complex. The combination of intercharge distance and non-polar region in a groove may be important in the specificity of binding of local amines and their receptors. For example heparins may vary slightly in the position or number of sulfate groups, while forming secondary structures which differ sufficiently in spacial relations to accommodate or exclude a given local hormone. Thus, the degree of sulfation at the (C-3)-OH position of the uronic acid moiety could regulate conformation and biological activity in this regard. ACKNOWLEDGEMENT
The authors wish to acknowledge the assistance of Mrs. L. G. CHILDERS in some of the spectrophotometric measurements. Biochim. Biophys. Acta, 136 (1967) 56 66
66
A. L. STONE, H. MOSS
REFERENCES I 2 3 4 5 6 7 8 9 IO 11 i2 13 14 15 16 17 18 I9 20 21 22 23 24 25 26 27 28 29 3o 31 32 33 34 35
H. MASAMUNE, M. SUZAKI AND Y. KONDOH, J. Biochem. Tokyo, 3 ~ (194 o) 343J. E. JORPES, H. BOSTROM AND V. MUTT, J. Biol. Chem., 183 (195 ° ) 607 . M. L. WOLFROM AND W. H. MCNEELY, J. Am. Chem. Soc., 67 (1945) 748. A. B. FOSTER AND A. J. HUGGARD, Advan. Carbohydrate Chem., i o (1955) 335. A. g . FOSTER, E. F. MARTLEW AND M. STACEY, Chem. Ind. London, (1953) 899. L. VELLUZ, G. NOMIND AND J. MATHIEU, Bull. SOC. Chim. Biol., 41 (1959) 415 . I. DANISmFSKY AND H. STEINER, Federation Proc., Abstr. No. 2297, 48t h A n n u a l M e e t i n g , Chicago, 1964 . 51. L. WOLFROM, T. R. VERCELLOTTI, H. TOMOMATSU AND D. J. HORTON, Org. Chem., 29 (I964) 54 ° . A. L. STONE, Biopolymers, 2 (1964) 315 . I. TINOCO, JR., J. Chem. Phys., 33 (196o) 1532. I. TINOCO, JR., R. W. WOODY AND D. F. BRADLEY, J. Chem. Phys., 38 (1963) 1.317 . D. F. BRADLEY, 1. TINOCO, JR. AND R. W. WOODY, Biopolymers, I (1963) 239. I. TINOCO, JR., J. Am. Chem. Soc., 86 (1964) 297. 1. TINOCO, JR. AND R. W. WOODY, J. Chem. Phys., 4 ° (1964 ) 16o. It. DEFOE, J. Chem. Phys., 43 (1965) 3199 . H. DEVOE, J. Chem. Phys., 4 i (1964) 393. A. L. STONE AND D. F. BRADLEY, to be p u b l i s h e d . .~. L. STONE, L. G. CHILDERS AND D. F. BRADLEY, Biopolymers, I (1963) 11 t. A. L. STONE, Biopolymers, 3 (1965) 617A. L. STONE AND D. F. BRADLEY, I38th Natl. Meeting Am. Chem. Soc., New York, ±96o. A. L. STONE AND D. F. BRADLEY, J. Am. Chem. Soc., 83 (1961) 3627 . A. L. STONE AND D. F. BRADLEY, to be p u b l i s h e d . D. N. HOLCOMB AND I. TINOCO, JR., Biopolymers, 3 (I965) 121. J. N. VOORNAKIS AND H. A. SCHERAGA, Biopo@mers, 4 (1966) 33. V. S. R. RAO AND J. F. FOSTER, J. Phys. Chem., 69 (1965) 636. C. J. BROWN, Acta Cryst., 4 (I95I) IOO. E. A. MEYERS ANn W. N. LlPCOMB, Acta Cryst., 8 (i955) 583. G. ('. PIMENTAL AND A. L. MCCLELLAN, The Hydrogen Bond, W. H. F r e e m a n , San F r a n c i s c o , 190o, pp. 285 293. D. M. NEVILLE, JR. AND D. F. BRADLEY, Biochim. Biophys. Acta, 5 ° (1961) 397J. S. BRIMACOMBE AND J. M. \VEBEER, Mucopolysaccharides, B.B.A. L i b r a r y , Vol. 6, E l s e v i e r , A n l s t e r d a m , 1964, p. lO 5. V. S. R. RAO AND J. F. FOSTER, Biopolymers, t (1963) 527 . V. S. R. RAO AYD J. F. FOSTER, Biopolymers, 3 (1965) 185. A. HYBL, R. E. RUNDLE AND D. E. WlLLIA,~lS, J. Am. Chem. Soc., 87 (1965) 2779. K. FREUNDE.~BERG, E. SCHAAF, G. DUMPERT AND T. I~LOETZ, Nalure, 27 (1939) 850. R. S. BEAR, J. Ant. Chem. Soc., 64 (~942) 1388.
Biochim. Biophys. Acta, I36 (1967) 56 66
BIOCHIMICA ET BIOPHYSICA ACTA
BBA 25 737 ANOMALOUS ROTATORY D I S P E R S I O N OF METACHROMATIC MUCOPOLYS A C C H A R I D E - D Y E COMPLEXES II. H E P A R I N - M E T H Y L E N E B L U E COMPLEXES AT ACIDIC p H AUI)I~EY L. STONE AND H E R B E R T MOSS* Laboratory of Neurochemislry, National Institule of )3enlal Health, Bethesda, Md. 2ooz 4 (U.S.A.) (Received J u l y I8th, I956)
SUMMARY
The phenomenon of induced optical rotatory dispersion (induced Cotton effect) in symmetric planar dyes when bound to certain asymmetric polyanions has been further investigated for methylene blue-heparin complexes in order to determine the source of the a s y m m e t r y in the bound dye aggregate. Induced rotation was measured as a function of p H in the range of ionization of the carboxyl group of heparin. Spectrophotometric titrations showed that at p H 2.4 essentially all the carboxyl groups were unionized while at p H 3.1 about 2o % of the carboxyl groups were negatively charged. The metachromasy of the stoichiometric complexes changed towards the orthochromatic color upon loss of the carboxyl-group binding site. Analysis of the metachromatic spectra according to quantitative parameters relating to dye-dye interactions showed, however, that although hypochromism (loss of oscillator strength) was decreased at p H 3.1 and 2.4 there was no concomitant decrease in the metachromatic shift in center of gravity. The rotatory dispersion curves were found to increase in magnitude with decreasing p H rather than to correlate solely with the decrease in dispersion interactions (hypochromism) or with the similarity of the degree of exciton-type dye-dye interactions (frequency shifts), showing the complex nature of the induced Cotton effect at p H 6. 7. The increase in magnitude of the effect from p H 6. 7 to 2.4 can be explained by the loss of a negative, dispersion-type Cotton effect which cancels negative and positive rotations of an exciton-type effect at p H 6. 7, or it could be the result of a conformational change in the asymmetric polymer-dye aggregate. The correlation of the source of the induced rotation at least in part with dye-dye rather than dye-polymer and/or dye-solvent interactions further supports the interpretation that the anionic sites of heparin in solution are in helical order. A molecular model of heparin is presented showing the helical order possible for c¢-(i-~ 4)-glucose polymers. The specific conformation is stabilized by a linear hydrogen bond involving the nitrogen of the sulfamino group as the acceptor and the C-3 hydroxyl group of the following uronic acid as the donor. Certain features of this molecular conformation are discussed in relation to the biological activity of heparin. * Visiting Chemist from High School Science Teacher Study-Work Institute, Summer, 1964
Biochim. Biophys. Acta, 136 (1967) 56-66
COTTON EFFECTS IN HEPARIN--METHYLENE BLUE COMPLEXES
57
INTRODUCTION
For over a decade secondary features of heparin structure have been suggested and alluded to in relation to its manifold biological activities even though knowledge of its primary structure was skeletal at the first proposals 1-6. With the rapid advance in understanding of the primary structure during the last few yearsT, s, such proposals still hold and m a y be approached in a more specific fashion. Two separate aspects of secondary structure possible for heparin are (i) helical order due to the ~ - ( I - ~ 4 ) D-Glc repeat configuration, analogous to the amylose structuree, 8 and (2) a linkage between the sulfamino function and an unsubstituted - C - O H , e.g., the "intramolecular sulfate bridges - N - S O 2 0 C - " (refs. I, 4, 5). In regard to the first feature, helical order for the anionic sites of heparin in solution was indicated b y the demonstration 9 that four, chemically different, planar symmetric dyes exhibit induced anomalous optical rotatory dispersion in the metachromatic absorption band when bound to this polyanion. It was shown that the induced Cotton effect was dependent upon the metachromatic reaction of the dyes and upon the integrity of the heparin molecule, particularly in regard to the sulfamino group. The data showed that the dyes must be bound, but in addition, that they must be bound to nearby sites, supporting the view that the induced a s y m m e t r y was largely dependent upon dye-dye rather than dye-polymer interactions. Induced Cotton effects have been predicted for chromophores in a helical array using various theoretical models 1° ,6. The exciton-coupling model '2,'3 predicts a non-symmetric rotatory dispersion curve with a dominant feature, the peak or trough, flanked by two minor troughs or peaks, respectively, giving a net change in rotation equal to zero. The coupling of different electronic bands among chromophores (dispersion interactions) results in a symmetric Cotton effect in the visible or ultraviolet absorption region which is compensated by an equal and opposite Cotton effect in a shorter-wavelength absorption band so that an apparent net rotation m a y occur in any one absorption band '2. The non-symmetric shape of the curve for the methylene blue-heparin complexes also supported the hypothesis that the induced rotation derives largely from dye-dye rather than dye-polymer interactions. Further, it was found that the addition of histamine to the complex caused a small increase in induced rotation. Hence the ordered array of dyes leading to the induced rotation was apparently not destroyed b y the addition of histamine to a site on the heparin molecule. Insofar as the role of the sulfamino group in the secondary structure of heparin is concerned, hydrolysis of about 33 % of the N-sulfates which is known 4, ~ to cause a loss of almost all the anticoagulant activity, led to a marked loss of the induced a s y m m e t r y in bound methylene blue with relatively slight loss of metachromasy. The data might be interpreted as indication of a change from a "native" molecular conformation if the source of the induced rotation were known in regard both to the pertinent binding sites and to the dye-dye interactions, since exciton-type interactions between neighboring dyes would be directly dependent upon intersite conformations. In the present study the role of the carboxyl group in the induced rotation of the methylene blue-heparin complex was investigated. The results showed that changes in metachromasy and induced rotation of the dye-aggregate (or polymer-site geometry) occur upon ionization of the carboxyl group which support the interpretaBiochim. Biophys. Acta, 136 (I967) 5 6 - 6 6
58
A . L . STONE, H. MOSS
tion that at least part of the induced rotation at neutral pH is due to dye-dye interaction and hence to a helical array of anionic sites. Furthermore, a molecular model of heparin was constructed, using Courtauld atomic models, which shows secondary structural features consistent with the induced anomalous rotation of the dye complexes. MATERIALS AND METHODS
The spectrophotometric techniques involving polymer-dye complexes were described previously 17-21. Samples of heparin and methylene blue were the same preparations as in the previous studies 9,17. Histamine was obtained as the dihydrochloride from Nutritional Biochemicals, Inc. The concentration of the dye and heparin anionic sites was about I. 4. lO.5 M at all pH values with absorbance less than I.O unless otherwise stated. Buffers were I mM potassium phthalate at pH 2.4 and 3.1 and I mM sodium cacodylate at pH 6. 7. Measurements of rotation were made on a Rudolph spectropolarimeter model 2oo using a symmetrical angle of 1.8, pathlength of 2.5 cm and fused quartz cells made by Opticett Co., Inc. Results with the Rudolph spectropolarimeter have yielded anomalous rotation dispersion curves with sharper troughs and smaller amplitudes than the Bendix spectropolarimeter 9, which was thought to be due to a narrower band pass in the former instrument. The optical parameters of the metachromatic reaction relative to those of the free dye were obtained as described previously ~2 from calculation of the oscillator strength (f), the center of gravity and the band half-width of the various spectra using program HJDoo8C (written by Dr. H. DEVOE) in the Honeywell 8oo computer. Molecular models were made with the Courtauld atomic models (Ealing Corp., Cambridge, Mass.) with the relative scale of o.8 incer per A. RESULTS
Induced anomalous rotatory dispersion curves for stoichiometric methylene blue-heparin complexes at pH 6. 7, 3.1 and 2.4 are shown in Fig. I. Stoichiometry based upon titration end points 17 indicated that about 80 % of the carboxyl groups were unionized at pH 3.1 and IOO % at pH 2. 4 under these conditions. It can be seen that relative to pH 6. 7 the induced rotation of the dye is not reduced when the carboxyl site is removed, but is increased and slightly displaced towards a higher wavelength. It is noteworthy that the three curves intersect at a common "isorotation" point, 566 m/x. Although the curves do not represent the carboxyl ionization per se, they represent a change in the structure of the methylene blue-heparin complex due to this ionization. A series of curves with a common non-zero point of rotation has been reported recently for the double-helix to single-chain-helix transition of a polynucleotide ~ and for the "stacked" to "unstacked" transition in single-strand trinucleotides ~4. Table I summarizes the values of the various optical parameters (see text in table). The oscillator strength is a measure of the total intensity of the absorption band while the center of gravity and the band half-width give the intensity-weighted average frequency and the frequency distribution of the band. Changes in these parameters as compared with those for the free dye are quantitative measures of the Biochim. Biophys. Acta, 136 (1967) 56-66
COTTON +10
EFFECTS
IN
HEPARIN--METHYLENE
I t I I I I I I I I I I I I I
BLUE
COMPLEXES
59
I J ~ I I I i r I I I
3 + 5--
/
~ 3.-w". . . .
~
~----t 2
- 5--
g ,%
~ -I0--15 --
-20 --
I _25 I I I I I ] I I I r I I f t P I 450 500 550 600 m~
I F I I I I I I I I I 650 700
I 750
F i g . I. I n d u c e d o p t i c a l r o t a r y d i s p e r s i o n o f m e t h y l e n e blue-heparin with ionization of carboxyl g r o u p s , q~ ~ m o l a r r o t a t i o n o f t h e d y e - - I o n / M / w h e r e M is t h e m o l a r i t y o f t h e d y e s o l u t i o n , the observed rotation and 1 the pathlength i n d e c i m e t e r s . C u r v e i a t p H 6 . 7 ; C u r v e 2 a t p H 3.1 ; C u r v e 3 a t p H 2. 4. T h e b r o k e n l i n e s i n d i c a t e i r r e g u l a r i t y in e x p e r i m e n t a l points.
degree of metachromasy 22, Acg (frequency shift) giving an estimate of the strength of the interaction between like transition moments of the bound dyes and Af (hypochromism) giving the extent of interaction between non-identical transition moments of the dyes, and solvent or dye and polymer. The ratio of absorption at 66o m/~ to that at 61o m~, the ~//3 ratio, which has often been used as a measure of metachromasy, is also included. However, recent evidence 22 shows that for most polymerdye systems this ratio does not parallel 2cg (or/If) in magnitude and therefore cannot be taken as a comparative measure of metachromasy from a theoretical viewpoint. The values of the spectral parameters of the free methylene blue (Table I) do not change with pH. It might be expected that metachromasy will decrease with a decrease in anionic site density and it can be seen t h a t / I f and A~/~ decrease as the carboxyl group site is lost, going toward the bluer, or orthochromatic color for the solution of the complex. However, A cg and ABW do not decrease. The former increases slightly at p H 3.1 while the latter is increased at p H 2.4 and 3.1. Thus, "decreased metachromasy" in this case consists of a loss of hypochromism without concomitant decrease in frequency shift. Addition of histamine to the complexes at p H 2.4 at ratios H i / H of 1. 7 or 5 caused a small decrease in all the metachromatic parameters. At a ratio of 22, however, greater decreases in metachromasy occurred in all the optical parameters with a relatively slight decrease in Oa. Comparison of the various parameters at the three p H values are made in the last 6 columns of Table I based upon the values of the p H 6.7 complex as IOO %. Biochim. Biophys. Acla, 1 3 6 ( I 9 6 7 ) 5 6 - 6 6
I
Do
h~
PARAMETERS
OPTICAL
OF METHYLENE
BLUE
AND
METHYLENE
BLUE
tIBPARIN
COMPLEXES
pH
6. 7 3-1 2. 4
6. 7 3.1 2.4
2. 4 2. 4 2. 4
Sample
MB MB MB
MB-H 3IB H MB-H
MB-H-Hi MB-H-Hi MB-H-Hi
1. 7 5.0 22.0
-
-
[5-1
[Hi]
o.133 o.13o O.ll 3
o.228 o.179 o. I5O o.81 o.76 o.51
o.84 0.89 0.83 °-42 o.41 0.30
o.26 0.38 0.39
A B IV
Acg
t .3 8
(£~/~Z 1 )f
BW
<'If
3)
1.33 i "35
i X IO
16.o6 15.99 15.97
(£m
cg
0.665 o.67o o.675
f I0
1.92 1.9o 1.88
c~/fl
o-35 o.32 o.18
o.74 0..56 o.42
A~I~
"3)
278 28o 253
i86 237 282
585 585 585
581 583 585
~ At~ (× zo ~) (mu)
5)5 5t5 545
(my)
2,p
1oo 74 66
58 57 5°
~oo 79 66
.4~ionic .l/ sites
47 43 22
Ioo 76 56
I~I~
61
97 90
IOO lo6 99
"Icg
161 155 i15
IOO 146 15o
149 151 ~36
IOO 12"~ 152
I B IV ~Pa
Per ce~t of resultams qf parameters (fill 6.7)
T h e o p t i c a l p a r a m e t e r s h e r e a r e for m e t h y l e n e b l u e - h e p a r i n c o m p l e x e s c l o s e t o P / D -- i w h e r e P / D is t h e r a t i o of a n i o n i c s i t e s t o d y e m o l e c u l e s . M e t a c h r o m a s y (./If, Acg, A B W ) in t h e m e t h y l e n e b l u e h e p a r i n s y s t e m is g r e a t e r a t h i g h e r r a t i o s (1>/19 = 2 4) a t t h e t h r e e p H v a l u e s . (.:.oncentratiol: of d y e s a n d a n i o n i c s i t e s for a l l t h e c o m p l e x e s is a b o u t 1. 4. 1o -5 M. O p t i c a l p a r a m e t e r s as p r e v i o u s l y r e p o r t e d j7 a r e s y m b o l i z e d a s f o l l o w s : f, o s c i l l a t o r s t r e n g t h ; cg, c e n t e r of g r a v i t y ; B\V, b a n d h a l f - w i d t h in I o -a r e c i p r o c a l c e n t i m e t e r s ; ~//~, t h e r a t i o of a b s o r b a n c e a t t h e ~ b a n d (665 lnjt) t o a t 3 s o r b a n c e a t t h e fl b a n d (61o m u) of t h e a b s o r p t i o n s p e c t r u m ; .If, .)cg, A B W ,
l
TABLE
©
;:u
bl
o
>
o
62
A . L . STONE, H. MOSS
Fig. 2. Views of a m o d e l of h e p a r i n c o n t a i n i n g two dimeric r e p e a t i n g units. (a) O u t s i d e s u r t a c e s h o w i n g spiralling p y r a n o s e o x y g e n s (p) a n d t h e c a r b o x y l a n d t h e (C-3)-O sulfate groups. T h e C-6 h y d r o x y l g r o u p of t h e g l u c o s a m i n e is p o i n t i n g t o w a r d s t h e reader. T h e O-sulfate g r o u p could be placed on t h e C-6 h y d r o x y l g r o u p i n s t e a d of t h e C-3. I n t h e model, t h e C-6 h y d r o x y l is clearly available for e t h e r linkage to protein. (b) Inside surface s h o w i n g t h e groove, spiralling glycosidic o x y g e n s (white dots), linear h y d r o g e n b o n d b e t w e e n t h e a m i n o s u g a r n i t r o g e n (acceptor) a n d t h e C-3 h y d r o x y l of t h e following uronie acid. T h e d i s t a n c e available in t h e groove b e t w e e n t h e Nsulfate a n d p r e c e d i n g c a r b o x y l g r o u p is a b o u t 7.5-8 ~k w h i c h can r e a d i l y a c c o m m o d a t e h i s t a m i n e with t h e far ring n i t r o g e n n e a r t h e c a r b o x y l a n d t h e a m i n e n i t r o g e n n e a r t h e sulfate.
Biochim. Biophys. Acta, I36 (1967) 5 6 - 6 6
COTTON EFFECTS IN HEPARIN--METHYLENE BLUE COMPLEXES
63
Molecular models Fig. 2 shows several views of a model of heparin based upon an a-(I --~ 4)glycosidic repeating linkage with the normal chair conformation in the sugar rings 25 and the 0-sulfate on C-3 of the glucosamine moiety. Placing the O-sulfate on C-6 tends to put this sulfate group more on the outside of the molecular contour (see Fig. 2a where the C H 2 0 H points toward the reader) but does not alter fundamental relations between the repeating units. As was shown previously by VELLUZ6 with molecular models of hexamesitylene-heparin complex, the heparin molecule can assume a helical or spiral form anticipated from the amylose-like repeating unitsT, s. There is some flexibility to the molecule, but a specific conformation would be stabilized b y the formation of a hydrogen bond between the C-3 hydroxyl of the glucuronic acid moiety and the nitrogen of the preceding glucosamine ring. This bond is readily formed in the model and appears to be the only linear hydrogen bond possible for the spiral conformation. Such - O H . . . N bonds have been described in the crystalline structures of several compounds, e.g., p-aminopheno126 and hydroxylamine 27, and appear to be reasonably strong, of the order of magnitude of hydrogen bonds which occur in amides and peptides 28. The views of the model (which consists of two dimeric units) show both the twisting of the glycosidic oxygens close to the axis of the molecule (dots) (Fig. 2b) and the spiralling of the pyranose oxygens (P's) along the helix (Fig. 2a). It can be seen that the negative binding sites for the cationic dyes (0-sulfate, N-sulfate and carboxyl) would impart a helical order to a bound dye aggregate, which could give rise to the observed induced Cotton effect(s). Furthelanore, one can readily place a model of the histamine molecule in the groove caused by the spiralling of the molecular surface. In this position the histamine can span the groove between the N-sulfate and the preceding carboxyl sites with its primary amine end at the N-sulfate and its ring nitrogen end near the carboxyl group. Furthermore, there are two axial hydrogens in the groove which constitute a small area of non-polarity which could interact with the imidazole ring. A model of serotonin could be seen to fit less readily than histamine in the heparin groove, since it is more bulky and over 2 A longer.
DISCUSSION
The phenomenon of induced anomalous rotation in methylene blue-heparin complexes could be interpreted relative to polymer conformation if it could be shown that the induced Cotton effect is due to dye-dye interactions among bound cationic dyes, which represents the spacial orientation of fixed substituents on the polymer chain. Previously 9 it was shown that: (I) the phenomenon is generally observed among chemically different cationic dyes bound to heparin, rather than peculiar to methylene blue; (2) it is a property of bound dyes rather than of the free dye in a solution of an optically active polymer since competitive inhibition of ~binding by bivalent ions diminished asymmetric metachromatic interactions; and finally (3) d y e dye interactions are involved since distribution of bound dyes among m a n y excess sites causes concomitant shifting and ultimate disappearance of the a s y m m e t r y and metachromasy which would not be expected for dye-polymer interactions alone. The present study shows that the induced rotation is due substantially to exciton-type Biochim. Biophys. Acta, 136 .(1967) 56-66
64
A. L. STONE, H. MOSS
dye-dye interaction which permits further interpretation of the induced rotation and conformational aspects of the polymer. In principle the induced rotations exhibited by dye-polymer complexes can be analyzed for their exciton, dispersion, and other possible components in a manner analogous with the separation of hypochromism and frequency shift. Such quantitative calculations are not made in this report because the magnitude of the error in rotations above 62o m/~ preclude the evaluation of an integrated value for rotation, i.e., rotatory strength. Measurements are in progress using a photocell more sensitive at the upper wavelengths. Qualitatively, however, irregularities in induced rotation curves near the trough or peak regions which have been observed in this and other systems18,19, 29 indicate the complex nature of the Cotton effects.
Hypochromism The Af (Table I) decreases by one-third as one out of the three sites available per dimeric sugar unit at p H 6. 7 is no longer occupied b y dye, showing that dispersiontype interaction occurs between dyes bound to the carboxyl sites and other dyes, the polymer and/or the solvent. Furthermore, the percentage in Af follows closely the percentage change in total anionic sites. The Cotton effect, however, is not reduced, showing lack of direct correlation between Af and the induced rotation and indicating further that the effect is the resultant of more than one type of interaction. If the dispersion interaction at p H 6. 7 gave rise to a negative Cotton effect in the visible band, its positive contribution would cause negative rotations on the lower-wavelength side of the resultant curve to be less than at p H 2,4, which is what is observed. The possible presence of a small number of L-iduronic acid residues in the primary structure of heparin 3° m a y prove to have bearing on the magnitudes of the resultant effects at the various p H values if dye-polylner interactions are important in the hypochromic effect.
The frequency shift It has been found12,13 that generally as the average number of chromophore units in an exciton interaction increases, both the frequency shift and the Cotton effect increase in magnitude, all other factors being equal. In this system, the 2cg might be expected to decrease from p H 6.7 to p H 2. 4 as the repeating number of bound dyes is reduced from 3 to 2 if all other factors remained the same. However, the Acg and the induced Cotton effect are not diminished while there is a decrease in Af supporting the argument that a substantial component of the Cotton effect is exciton induced and that other factors in addition to the average distance between the bound dyes are involved. Since the angular positions of the dyes in the aggregates (geometric factors) are also paramount in the net electronic interactions, the data suggest that the local neighboring dye-polymer conformations differ in the acid and neutral complexes. The change in the asymmetric metachromasy with p H might, therefore represent a conformational change in heparin rather than solely a change in the relative proportions of the various component parts of the resultant Cotton effect. L-Iduronic acid moieties 3° would be expected to affect the average geometric factors at p H 6. 7 and contribute to the observed changes with pH. However, such moieties, if present, would constitute a minor component of the primary structure. Biochim. Biophys. Acta, 136 (1967) 56-66
COTTON EFFECTS IN HEPARIN-METHYLENE BLUE COMPLEXES
65
Recent studies with amylose and carboxymethylamylose31,32indicate that these ~-(I~4)-glucose polymers undergo conformational changes in solution with change in pH. Carboxymethylamylose undergoes changes upon ionization of the carboxy! group (pH 2-6) which is believed to represent a helix-to-coil transition 32, however, amylose is believed to have helical order in both acid and neutral pH. The fact that there is an induced Cotton effect for methylene blue-heparin at pH 6. 7 indicates that some helical order exists among the sites in the heparin molecule at that pH as well as at pH 2.4. If at neutral pH the helical nature is partial or segmented as proposed for amylose 31, then the transition corresponding to that in CM-cellulose might be to a more rigid type of helix. The results of the addition of histamine to methylene blue-heparin complexes at pH 2.4 indicate that at ratios of Hi/H of about 2 and 5 little dye is displaced from sites available in the absence of histamine since only slight effects were seen in metachromasy and induced rotation. Studies on the extent of binding of histamine in this system would be necessary before any further conclusions could be reached. Molecular model In the past, secondary structural features have been ascribed to the N-sulfate group in terms of a "sulfate bridge"l,4, 5, -N-SO2-O-C-. The available chemical data, however, showed that all the sulfates were titratable 4,5. The present model (Fig. 2) proposes that the N-sulfate grouping is involved in the secondary structure of the molecule through hydrogen bonds between the amino nitrogen (aeceptor) and the C-3 hydroxyl of the following uronic acid. Molecular models show this structure to be readily formed and such O - H . . . N bonds have been described and can be reasonably strong 2s. Recent crystallographic analysis of the cyclohexaamylosepotassium acetate complex 33, which would be analogous to one turn of an amylose helix, shows a hydrogen bond between the (C-2)-0 and (C'-3)-O of the next sugar ring. This corresponds with the (C-2)-N... H-O-(C'-3) bond proposed above. Molecular models of polysaccharides show small areas of non-polarity which result from the twisting of the sugar rings relative to one another. In the case of fi-linked polymers such as the chondroitin sulfates A and C (where hydrogen bonds can also be formed, i.e., (C-3)-O-H... N-(C'-2) this region includes four C-H groups. The concept of non-polar regions in polysaccharide structure was brought out by FREUNDENBERG~4 (and discussed by BEAR35) from models of amylose. He considered the amylose molecule to be a helix with a center essentially "hydrocarbon" in nature accounting for the adsorption of 12 in the amylose-I 2 complex. The combination of intercharge distance and non-polar region in a groove may be important in the specificity of binding of local amines and their receptors. For example heparins may vary slightly in the position or number of sulfate groups, while forming secondary structures which differ sufficiently in spacial relations to accommodate or exclude a given local hormone. Thus, the degree of sulfation at the (C-3)-OH position of the uronic acid moiety could regulate conformation and biological activity in this regard. ACKNOWLEDGEMENT
The authors wish to acknowledge the assistance of Mrs. L. G. CHILDERS in some of the spectrophotometric measurements. Biochim. Biophys. Acta, 136 (1967) 56 66
66
A. L. STONE, H. MOSS
REFERENCES I 2 3 4 5 6 7 8 9 IO 11 i2 13 14 15 16 17 18 I9 20 21 22 23 24 25 26 27 28 29 3o 31 32 33 34 35
H. MASAMUNE, M. SUZAKI AND Y. KONDOH, J. Biochem. Tokyo, 3 ~ (194 o) 343J. E. JORPES, H. BOSTROM AND V. MUTT, J. Biol. Chem., 183 (195 ° ) 607 . M. L. WOLFROM AND W. H. MCNEELY, J. Am. Chem. Soc., 67 (1945) 748. A. B. FOSTER AND A. J. HUGGARD, Advan. Carbohydrate Chem., i o (1955) 335. A. g . FOSTER, E. F. MARTLEW AND M. STACEY, Chem. Ind. London, (1953) 899. L. VELLUZ, G. NOMIND AND J. MATHIEU, Bull. SOC. Chim. Biol., 41 (1959) 415 . I. DANISmFSKY AND H. STEINER, Federation Proc., Abstr. No. 2297, 48t h A n n u a l M e e t i n g , Chicago, 1964 . 51. L. WOLFROM, T. R. VERCELLOTTI, H. TOMOMATSU AND D. J. HORTON, Org. Chem., 29 (I964) 54 ° . A. L. STONE, Biopolymers, 2 (1964) 315 . I. TINOCO, JR., J. Chem. Phys., 33 (196o) 1532. I. TINOCO, JR., R. W. WOODY AND D. F. BRADLEY, J. Chem. Phys., 38 (1963) 1.317 . D. F. BRADLEY, 1. TINOCO, JR. AND R. W. WOODY, Biopolymers, I (1963) 239. I. TINOCO, JR., J. Am. Chem. Soc., 86 (1964) 297. 1. TINOCO, JR. AND R. W. WOODY, J. Chem. Phys., 4 ° (1964 ) 16o. It. DEFOE, J. Chem. Phys., 43 (1965) 3199 . H. DEVOE, J. Chem. Phys., 4 i (1964) 393. A. L. STONE AND D. F. BRADLEY, to be p u b l i s h e d . .~. L. STONE, L. G. CHILDERS AND D. F. BRADLEY, Biopolymers, I (1963) 11 t. A. L. STONE, Biopolymers, 3 (1965) 617A. L. STONE AND D. F. BRADLEY, I38th Natl. Meeting Am. Chem. Soc., New York, ±96o. A. L. STONE AND D. F. BRADLEY, J. Am. Chem. Soc., 83 (1961) 3627 . A. L. STONE AND D. F. BRADLEY, to be p u b l i s h e d . D. N. HOLCOMB AND I. TINOCO, JR., Biopolymers, 3 (I965) 121. J. N. VOORNAKIS AND H. A. SCHERAGA, Biopo@mers, 4 (1966) 33. V. S. R. RAO AND J. F. FOSTER, J. Phys. Chem., 69 (1965) 636. C. J. BROWN, Acta Cryst., 4 (I95I) IOO. E. A. MEYERS ANn W. N. LlPCOMB, Acta Cryst., 8 (i955) 583. G. ('. PIMENTAL AND A. L. MCCLELLAN, The Hydrogen Bond, W. H. F r e e m a n , San F r a n c i s c o , 190o, pp. 285 293. D. M. NEVILLE, JR. AND D. F. BRADLEY, Biochim. Biophys. Acta, 5 ° (1961) 397J. S. BRIMACOMBE AND J. M. \VEBEER, Mucopolysaccharides, B.B.A. L i b r a r y , Vol. 6, E l s e v i e r , A n l s t e r d a m , 1964, p. lO 5. V. S. R. RAO AND J. F. FOSTER, Biopolymers, t (1963) 527 . V. S. R. RAO AYD J. F. FOSTER, Biopolymers, 3 (1965) 185. A. HYBL, R. E. RUNDLE AND D. E. WlLLIA,~lS, J. Am. Chem. Soc., 87 (1965) 2779. K. FREUNDE.~BERG, E. SCHAAF, G. DUMPERT AND T. I~LOETZ, Nalure, 27 (1939) 850. R. S. BEAR, J. Ant. Chem. Soc., 64 (~942) 1388.
Biochim. Biophys. Acta, I36 (1967) 56 66