Induced cotton effects of hyaluronic acid-acridine orange complex and conformation of the polymer

BIOCHEMICAL

Vol. 52, No. 4, 1973

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INDUCED COTTON EFFECTS OF HYALURONIC ACIDACRIDINE ORANGE COMPLEX AND CONFORMATION OF THE POLYMER B. Chakrabarti and E.A. Balazs Department of Connective Tissue Research Boston Biomedical Research Institute 20 Staniford Street, Boston, Mass. 02114 Received

April

24,

1973

Summary: Hyaluronic acid-acridine orange complexes in water show a symmetric doublet CD band centered at 455 nm. The exciton-like CD band indicates a preferred left-handed chirality for this polymer in solution, which correlates with the proposed lefthanded double helical structure of hyaluronic acid in oriented film. The sign and general shape of the induced CD band with hyaluronic acid (dye concentration 2.5~10~5M) is the same throughout the range of polymer-dye ratio studied. The single CD band which appears at a lo-4M concentration of dye has been attributed to dye-dye interaction. Hyaluronic acid, preheated to 95“C for one hour, followed by rapid cooling, does not show any induced CD band. The remarkable conformational versatility of hyaluronic acid (HA) both in solution and solid form has recently been illustrated The sigmoidal change in molar rotation and ellipticity (1,2,3). with change in temperature and pH has been attributed to orderdisorder studies helical

transition

of hyaluronic

(1,2) have further structure of this

acid

(3,4).

X-Ray

diffraction

revealed the existence of a double polymer in oriented films.

Circular dichroism (CD) of acridine orange (AO) induced by binding to hyaluronic acid seems to be a suitable method for studying the basic aspects of the conformation of this polymer, since dye binding to asymmetric helical polymers such as nucleic acids (5,6) and polyglutamic acid (7) is well documented. The scope and applicability of the method in acidic polysaccharides have

also been assessed (8,9,10). In the present communication

we describe

the

induced

circular

dichroism and salt

of HA-A0 complexes at various polymer-dye ratios, pHs concentrations. Attempts have also been made to use hyaluronic acid denatured by heating at 95'C. Materials and Methods: Na-salts of hyaluronic acid prepared from human umbilical cord (according to a method previously described 1111) were obtained from Biotrics, Acridine orange was a commercial Copyright 0 I9 73 b-v Academic Press, Inc. AN rights of‘reproductiorz in atzv jbrm reserved.

Inc., Arlington, Mass. sample purified according 1170

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Stone and Rradley (12). Na-hyaluronate was dialyzed against distilled water before use and the pH of the dialyzed solution was adjusted to approximately 7.0. The dye concentration for complex formation was maintained at 2.5~10~'M throughout the experiments, except when indicated otherwise. It has been found that the manner of mixing dye and hyaluronic acid influences the optical properties and solubility of the complex. For example, when acridine orange solution is added to a concentrated polymer solution, precipitation may occur; whereas a concentrated solution of hyaluronic acid could be added to a dilute dye solution without any resulting precipitation, The latter method was chosen for the preparation of all solutions. Optical measurements were carried out with a Cary Model 15 Recording Spectrophotometer (absorption spectra) and a Jasco-5 spectropolarimeter (circular dichroism). Fresh solutions were used and measured immediately after mixing. The c.oncentration of hyaluronic acid is expressed in terms of the average molecular weight of the disaccharide repeating unit. The values of molar ellipticity are calculated on the basis of dye concentration and are uncorrected for the refractive index of water. Figure 1 shows the induced symmetric Results and Discussion: doublet CD band of a solution in which the molar ratio of the The hyaluronic acid disaccharide and the acridine orange was one. curve has a positive peak at 480 nm and a negative peak at 430 nm. The spectrum corresponds with that of a left-handed exciton band centered at 455 nm. The CD parameters of solutions containing different molar ratios of the polymer and dye (P/D) are listed in Table I. It can be seen that the sign and general shape of the curves remain the same when the dye concentration is 10 -5M . With increasing dye concentration (5x10 -4W > a single CD band appears (Fig. 1). The metachromatic absorption bands at different P/D values are shown in Fig. 2. The observed hypochromism in the dye band with addition of HA to A0 is not accompanied by any shift of the cx band at 492 nm, but the ratio of the intensity of the c1 and B bands (465 nm) is different at different P/D values. Moreover, at low The ratio of c( and B bands is P/D, the y band (450 nm) appears. close to one when P/D is in the range of 0.70 to 1.2 and only at this range can a symmetric CD doublet be observed. The significance of this -relationship is not presently understood. 1171

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Fig.

1.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Circular dichroism band of HA-A0 complex. a. P/D = 1.02; dye concentration 2.5~1O-~M b. 5x10-4M P/D = 0.95; dye concentration C. Same P/D and dye concentration as,in (a) but hyaluranic acid was preheated at 95O for one hour and slowly cooled down to room temperature Optical path length 3.5 cm.

When the concentration

sodium chloride (pH 5.0 to 1.5)

(10 -4M to 10 -1 M) or the hydrogen ion is increased in a solution with P/D

near one, the magnitude of the CD doublet decreases without any change in sign and shape of the curve. At a salt concentration of 0.05M and pH 3.5, the spectrum almost disappears. cooled

When hyaluronic acid, to +l°C and brought

heated at 95°C for to room temperature

one hour, was rapidly and then used for

induced Cotton effect measurements, no doublet CD could be found. Whereas, with hyaluronic acid cooled slowly (over a one hour period) to room temperature, a doublet CD band, with considerably less intensity, was found. Stryer and Blout (7) have shown that a symmetric dye, such as acridine orange, exhibits optical activity when bound to asymmetric helical polyglutamate. The dye shows not optical activity when bound to polyglutamate which is in random coil form. Furthermore, the induced Cotton effects were found to be dependent upon the chirality of the helix (7). Metachromatic reaction and induced Cotton effects have been predicted for chromophores using various theories such as exciton-dispersion and classical oscillator models (13,14). Adjacent dye molecules, when fixed in parallel

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

1 FPEE

A0

2 P/D

IO

0

A ,nm Fig.

2.

Metachromatic uranic acid 1 cm.

planes perpendicular among like transition band

appears

relative certained

absorption at different

of acridine orange P/D. Optical path

with hyallength

to the polymer axis, oscillators in the

when there

is

a helical

undergo optical doupling long axis. A doublet CD translation for each dye

to its neighbor (15). The sense of helix from the peaks of this doublet CD band;

can also a negative

be aspeak

at the high frequency and an equal positive peak at lower frequency indicate left-handedness and the converse is true for right-handedness (15). Dea et al (1) have studies that in oriented films a double helical are antiparallel of the symmetric

shown from their x-ray diffraction of hyaluronic acid, there can exist

form in which two identical left-handed strands to one another. The observed left-handed chirality CD doublet for the HA-A0 complex is consistent

with

the A0 ference despite

proposed (1) left-handed double helix. has been considered (9) a poor dye for any structural inunless the modes of binding have been adequately assessed, the wide use of this dye for structural studies of DNA (5, In our previous work (3), it has been pointed 6) and RNA (16,17). out that in solution certain helical junction zones probably exist in the domain of the hyaluronic acid chain. The disappearance of

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I CD Parameters

Dye cont.

5

P/D+

10 5.3 2.2 1.3 1.02 0.81 0.52 0.32 *

of HA-A0 Complexes*

5 x10 -4

of

the

h2 X10

1.1 1.6 2.4 3.9 6.7 6.0 3.2 2.1

+480 +480 +480 +480 +480 +480 +475 +473

X1 and X2 are the wavelengths

Ac

457 457 455 455 455 454 453 452

CD peaks;

crossover wavelength; O1 and 0, are molar CD peaks at X1 and AZ, respectively.

-428 -430 -430 -430 -430 -430 -428 -428

Ac is

0.82 1.4 2.6 4.2 7.5 6.8 3.0 1.5

the

ellipticities

+Polymer concentration was calculated on the basis acid determination according to a method described

0, -4

zero

of the

of hexuronic previously

(20) *

exciton-like plained

CD band with on the basis

higher

of partial

and non-resonance type bound dye redistributes

or lower helical

P/D values character

of interaction. With itself to relatively

can be ex-

of the polymer

excess polymer, the more non-asymmetric

regions of the polymer chain (9) minimizing the doublet CD band. The finding is comparable to the recently reported results (18) with the methylene blue-carrageenan complexes, in which a reversal of sign of quantitative

the CD doublet with high P/D has been interpreted increase of the left-handed A carrageenan-like

as the portions

in the right-handed K form. In the case of HA, we think that in the presence of excess dye, non-resonance interaction (aye-aye or dye-polymer) causes the disappearance of induced CD bands. The single CD (Fig. 1) which appears only at higher concentration A0 (-5~10-~ M) can be attributed to asymmetric stacking of the

of dye.

Heat denatured hyaluronic acid, when rapdily cooled, does not show any induced Cotton effects, but the annealed one still exhibits some. Both of these show a reduced ultraviolet CD and ORD value with respect to the undenatured hyaluronic acid (B. Chakrabarti

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and E.A. Balazs, unpublished results). It seems reasonable to assume that there is a difference in the amount of helical junction points between the two HA preparations: most probably the rapidly cooled one having none. Our finding of the effect of pH and salts on the CD of HA-A0 complexes is in accord with the finding of Cleland's (19) ORD studies of this system. The metachromatic reaction disappears when the NaCl or hydrogen ion concentration is increased. Since the polymer-dye binding is primarily Coulombic in nature, the ion exchange competition of H+ or Na + with the AO+ for available negative sites of the polyanion seems to be the explanation for this phenomenon. Acknowledgement: This work was supported by USPHS grant (2ROl EY00223-11Al). 1.

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