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The structure of heparin: Interaction of heparin with basic dyes by visible spectrometry
646
SHORT COMMIJNICATIONS
BBA 23528
The structure of heparin: Interaction of heparin with basic dyes by visible
spectrometry* Heparin is a mucopolysaccharide consisting of partially sulfated units of =-D-glucuronic acid and 2-amino-2-deoxy-=-glucose joined by 1,4-bonds. Structurally, heparin can be treated as a polyanion type of polyelectrolyte with Na +, Li + or Ba 2+ as counterions. It is generally believed1, z that within the tetrasaccharide units there are 7 anions consisting of 2 carboxylic groups from the uronic portion, 2 Nsulfamino groups from the hexoamine portion and 30-sulfo groups on the hydroxyls of both sugar portions. In order to elucidate further the fine structure of heparin, four basic dyes, namely, Azure A (Allied), methylene blue (Eastman), basic fuchsin (Matheson, Coleman and Bell) and brilliant cresyl blue (Allied), were allowed to react with heparin in distilled water. In all cases the concentration of the basic dye was fixed, so varying quantities of heparin from a concentration range of/~g/ml to dg/ml were allowed to interact. In all cases examined, the maximum absorption of the basic dye was found to be at a long wavelength (Azure A, 6350 A; methylene blue, 6700 A ; basic fuchsin, 5600 A; brilliant cresyl blue, 625 ° 2~). With a trace amount of heparin added, a shorter wavelength band is formed. Increasing the heparin concentration increases the intensity of the shorter wavelength band and decreases the longer wavelength dye absorption band until the dye band reaches a minimum. Further increase of heparin concentration causes the intensity of the long-wavelength band to increase until a higher concentration is reached at which the intensity of the short wavelength band is diminished and the original dye band reappears. The maximum of the shortwavelength band is dependent on the heparin concentration; the energy corresponding to the absorption wavelength decreases as heparin concentration increases. However,
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ENERGY OF ABSORPTION, MAXIMA (cm -1 ) x 10 -3
Fig. I. Dependence of h e p a r i n concentration on a b s o r p t i o n m a x i m a . The dye concentration r e m a i n s c o n s t a n t t h r o u g h o u t the experiment, the values are listed in Table I. • This p a p e r represents one p h a s e of research performed for the J e t Propulsion L a b o r a t o r y , California I n s t i t u t e of Technology, sponsored b y the National Aeronautics and Space Administration, Contract NAS7-IOO.
Biochim. Biophys. Acga, 184 (1969) 646-648
647
S H O R T COMMUNICATIONS
there is a noticeable change in the slope if heparin concentration is still higher (Fig. I). When the intensity ratio of the short-wavelength band and the long-wavelength band are plotted v e r s u s the heparin concentrations, a bell-shaped distribution pattern is obtained with a maximum showing that the long-wavelength band is at a minimum in presence of a fixed concentration level of heparin (Fig. 2). I
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atN, ~° 1 • 10 -6
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,~/
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I
NH I"I
'l
a
a
0 I •
10 -7
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I 2
I 1
I 2
I 3
i~/,o F i g . 2. I n i t i a l r a t e o f t h e a d e n y l c y c l a s e r e a c t i o n . activity are given in the text. Protein concentrations:
Conditions
of the
measurement
of enzyme
i , 11. 7 m g / m l ; 2, 5.85 m g / m l ; 3, 2.92 m g / m l .
We interpret these facts as due to two processes. At a lower heparin concentration level, a complex is formed between the cation of the basic dye (D +) and the polyanions of the heparin (H-). The shorter wavelength band may represent the complex formed from the two ions. The ranges from the lowest concentrations of heparin to the maximum point (ab, Fig. 2) must represent the equilibrium between the dye positive ions and the complexes. At those maxima, the amount of complex so formed is highest at the expance of the dye. At this point (b, Fig. 2), the heparin held by the dye should correspond to an equivalent amount common to aU cation-containing substances. Within this range, different ratios of the two species will pass through an isosbestic point and this is the basis of the colorimetric method developed for the assaying of heparin by means of Azure A (refs. 3 and 4). For the second process, however, at still higher concentration levels, another new species is formed (see bc, Fig. 2). The new species seems to absorb at the same wavelength as that of the dye cation. Many dyes give the same electronic spectra Biochim. Biophys. Acta, 184 (1969) 6 4 6 - 6 4 8
648
SHORT COMMUNICATIONS
regardless of states of anion or cation 5. We postulate a consecutive reaction sequence D+C1 - + H - N a + --~ D + H - + NaC1 D + H - + H - N a + --~ D H y - N a +
This mechanism is based on two observations: (a) there are two different slopes for the two processes and (b) the intermediate species has a maximum and the optical intensity of the initial and final species vary exponentially with concentration. Evidence for the D+H- species is derived from the following facts: for the four basic dyes used, the heparin equivalent concentrations, based on the molecular weight of the basic dye used, yield an equivalent ratio about 0.5 as shown in Table I. These facts support the accepted structure of heparin 1,*. 2 dye molecules interact with each tetrasaccharide unit, the most alkaline groups reacting first. Apparently the reaction occurs preferably between 2 carboxylic groups or the 2 sulfoamino groups. This is consistent with the findings that basic dyes interact with nonsulfo-containing anions such as carboxylate, silicate, etc.s. TABLE I EQUIVALENT RATIO OF BASIC DYES WITH HEPARIN
Dye
Mol. wt.
Equivalent conch. (izg/ml)
Dye Cohen. (l~g/ml)
Equiv. wt.
Equiv. ratio *
Azure A M e t h y l e n e blue Basic fuchsin B r i l l i a n t cresyl blue
291.8 373.9 337 332.5
9.o 7 .o 8.0 25
4.1 3.6 3.3 9.7
642 725 808 856
0.52 0.59 0.66 0.69
• T h e o r e t i c a l v a l u e of i n t e r a c t i o n of 2 d y e m o l e c u l e s w i t h I t e t r a s a c c h a r i d e u n i t of h e p a r i n (1228) is o.5o.
We would like to point out that metachromasia does not depend to any significant extent on the polymerization of the dye by its aggregation 7 but depends on the sites of cations available for interaction. Linearity of the second process (bc, Fig. 2) clearly demonstrates this. It is believed that additional information will be gained by studying the interaction of polycations, such as cyclic diammonium salts, with polyanions and these investigations are now in progress.
Department of Chemistry, California State College, Los Angeles, Calif. 90032 (U.S.A.), and * Jet Propulsion Laboratory, California Institute of Technology, Pasadena, Calif. 9Ho3 (U.S.A.)
TEH FV YEN MASSOVD DAVAR ALAN REMBAUM
I L. B. JAQUES, L. W. KAVANAGH,M. MAZUEEK AND A. S. PERLIN, Biochem. Biophys. Res. Commun., 24 (1966) 447. 2 M. L. WOLFROM, J. R. VERCELLOTTI AND D. HORTON, J. Org. Chem., 29 (1964) 59o. 3 L. B. JAQUES AND A. WOLLIN, Can. J. Physiol. Pharm., 45 (1967) 787 • 4 F. C. MACINToSh, Biochem. J., 35 (1941) 776. 5 H. H. JAFFE AND M. ORCHIN, Theory and Applications of Ultraviolet Spectroscopy, J o h n Wi l e y, N e w Yo rk , 1962. 6 K. W. WALTON AND C. R. RICKETTS, Brit. J. Exptl. Pathol., 35 (1954) 227. 7 L. MICHAELIS, Cold Spring Harbor Syrup. Quant. Biol., 12 (1947) 131.
Received April 8th, 1969 Biochim. Biophys. Acta, 184 (1969) 646-648
SHORT COMMIJNICATIONS
BBA 23528
The structure of heparin: Interaction of heparin with basic dyes by visible
spectrometry* Heparin is a mucopolysaccharide consisting of partially sulfated units of =-D-glucuronic acid and 2-amino-2-deoxy-=-glucose joined by 1,4-bonds. Structurally, heparin can be treated as a polyanion type of polyelectrolyte with Na +, Li + or Ba 2+ as counterions. It is generally believed1, z that within the tetrasaccharide units there are 7 anions consisting of 2 carboxylic groups from the uronic portion, 2 Nsulfamino groups from the hexoamine portion and 30-sulfo groups on the hydroxyls of both sugar portions. In order to elucidate further the fine structure of heparin, four basic dyes, namely, Azure A (Allied), methylene blue (Eastman), basic fuchsin (Matheson, Coleman and Bell) and brilliant cresyl blue (Allied), were allowed to react with heparin in distilled water. In all cases the concentration of the basic dye was fixed, so varying quantities of heparin from a concentration range of/~g/ml to dg/ml were allowed to interact. In all cases examined, the maximum absorption of the basic dye was found to be at a long wavelength (Azure A, 6350 A; methylene blue, 6700 A ; basic fuchsin, 5600 A; brilliant cresyl blue, 625 ° 2~). With a trace amount of heparin added, a shorter wavelength band is formed. Increasing the heparin concentration increases the intensity of the shorter wavelength band and decreases the longer wavelength dye absorption band until the dye band reaches a minimum. Further increase of heparin concentration causes the intensity of the long-wavelength band to increase until a higher concentration is reached at which the intensity of the short wavelength band is diminished and the original dye band reappears. The maximum of the shortwavelength band is dependent on the heparin concentration; the energy corresponding to the absorption wavelength decreases as heparin concentration increases. However,
'"
10-1 ]
[
l
10 -2
I ~.
~\
!
I
I
I
r
"~
0
i0 -4
0'~
\
"Q ~.
"r" h o 1 • l0 -5
\ \
\~ ~0~
Azure A \
2
o u
I
r
x . "0 ~. METHYLENE OX\BLUE
J~
-3J
I
~
• lo"~
\ ,
, • ~0-'
i
16 000 16 500
'
17 000
,7'~00
~
18 000
I
18 500
I
g
000
?, 50020
000
ENERGY OF ABSORPTION, MAXIMA (cm -1 ) x 10 -3
Fig. I. Dependence of h e p a r i n concentration on a b s o r p t i o n m a x i m a . The dye concentration r e m a i n s c o n s t a n t t h r o u g h o u t the experiment, the values are listed in Table I. • This p a p e r represents one p h a s e of research performed for the J e t Propulsion L a b o r a t o r y , California I n s t i t u t e of Technology, sponsored b y the National Aeronautics and Space Administration, Contract NAS7-IOO.
Biochim. Biophys. Acga, 184 (1969) 646-648
647
S H O R T COMMUNICATIONS
there is a noticeable change in the slope if heparin concentration is still higher (Fig. I). When the intensity ratio of the short-wavelength band and the long-wavelength band are plotted v e r s u s the heparin concentrations, a bell-shaped distribution pattern is obtained with a maximum showing that the long-wavelength band is at a minimum in presence of a fixed concentration level of heparin (Fig. 2). I
I
I
c
10 -2
\o
\ 10 -3
%
\
\
10 -4
\
® ""2]c,O~ 10 -5 i
Azure
A
-
~'~'
10"6
E
a 10-7 .,~ ....
E -r I •
10 -2
I *
10 -3
I °
10 -4
I
I
I
I
I •
I
I
..2
J
I
t
/ c%
/ °,oL
I Ir
"~L
BRILUANT ~0 CRESYL BLUE
9
\
\
I • 10 -5
O/ /
atN, ~° 1 • 10 -6
" 2/
? , N"21 %% J
,~/
|
I
NH I"I
'l
a
a
0 I •
10 -7
I 1
I 2
I 1
I 2
I 3
i~/,o F i g . 2. I n i t i a l r a t e o f t h e a d e n y l c y c l a s e r e a c t i o n . activity are given in the text. Protein concentrations:
Conditions
of the
measurement
of enzyme
i , 11. 7 m g / m l ; 2, 5.85 m g / m l ; 3, 2.92 m g / m l .
We interpret these facts as due to two processes. At a lower heparin concentration level, a complex is formed between the cation of the basic dye (D +) and the polyanions of the heparin (H-). The shorter wavelength band may represent the complex formed from the two ions. The ranges from the lowest concentrations of heparin to the maximum point (ab, Fig. 2) must represent the equilibrium between the dye positive ions and the complexes. At those maxima, the amount of complex so formed is highest at the expance of the dye. At this point (b, Fig. 2), the heparin held by the dye should correspond to an equivalent amount common to aU cation-containing substances. Within this range, different ratios of the two species will pass through an isosbestic point and this is the basis of the colorimetric method developed for the assaying of heparin by means of Azure A (refs. 3 and 4). For the second process, however, at still higher concentration levels, another new species is formed (see bc, Fig. 2). The new species seems to absorb at the same wavelength as that of the dye cation. Many dyes give the same electronic spectra Biochim. Biophys. Acta, 184 (1969) 6 4 6 - 6 4 8
648
SHORT COMMUNICATIONS
regardless of states of anion or cation 5. We postulate a consecutive reaction sequence D+C1 - + H - N a + --~ D + H - + NaC1 D + H - + H - N a + --~ D H y - N a +
This mechanism is based on two observations: (a) there are two different slopes for the two processes and (b) the intermediate species has a maximum and the optical intensity of the initial and final species vary exponentially with concentration. Evidence for the D+H- species is derived from the following facts: for the four basic dyes used, the heparin equivalent concentrations, based on the molecular weight of the basic dye used, yield an equivalent ratio about 0.5 as shown in Table I. These facts support the accepted structure of heparin 1,*. 2 dye molecules interact with each tetrasaccharide unit, the most alkaline groups reacting first. Apparently the reaction occurs preferably between 2 carboxylic groups or the 2 sulfoamino groups. This is consistent with the findings that basic dyes interact with nonsulfo-containing anions such as carboxylate, silicate, etc.s. TABLE I EQUIVALENT RATIO OF BASIC DYES WITH HEPARIN
Dye
Mol. wt.
Equivalent conch. (izg/ml)
Dye Cohen. (l~g/ml)
Equiv. wt.
Equiv. ratio *
Azure A M e t h y l e n e blue Basic fuchsin B r i l l i a n t cresyl blue
291.8 373.9 337 332.5
9.o 7 .o 8.0 25
4.1 3.6 3.3 9.7
642 725 808 856
0.52 0.59 0.66 0.69
• T h e o r e t i c a l v a l u e of i n t e r a c t i o n of 2 d y e m o l e c u l e s w i t h I t e t r a s a c c h a r i d e u n i t of h e p a r i n (1228) is o.5o.
We would like to point out that metachromasia does not depend to any significant extent on the polymerization of the dye by its aggregation 7 but depends on the sites of cations available for interaction. Linearity of the second process (bc, Fig. 2) clearly demonstrates this. It is believed that additional information will be gained by studying the interaction of polycations, such as cyclic diammonium salts, with polyanions and these investigations are now in progress.
Department of Chemistry, California State College, Los Angeles, Calif. 90032 (U.S.A.), and * Jet Propulsion Laboratory, California Institute of Technology, Pasadena, Calif. 9Ho3 (U.S.A.)
TEH FV YEN MASSOVD DAVAR ALAN REMBAUM
I L. B. JAQUES, L. W. KAVANAGH,M. MAZUEEK AND A. S. PERLIN, Biochem. Biophys. Res. Commun., 24 (1966) 447. 2 M. L. WOLFROM, J. R. VERCELLOTTI AND D. HORTON, J. Org. Chem., 29 (1964) 59o. 3 L. B. JAQUES AND A. WOLLIN, Can. J. Physiol. Pharm., 45 (1967) 787 • 4 F. C. MACINToSh, Biochem. J., 35 (1941) 776. 5 H. H. JAFFE AND M. ORCHIN, Theory and Applications of Ultraviolet Spectroscopy, J o h n Wi l e y, N e w Yo rk , 1962. 6 K. W. WALTON AND C. R. RICKETTS, Brit. J. Exptl. Pathol., 35 (1954) 227. 7 L. MICHAELIS, Cold Spring Harbor Syrup. Quant. Biol., 12 (1947) 131.
Received April 8th, 1969 Biochim. Biophys. Acta, 184 (1969) 646-648