1907
EEFECTS OF ENVIRONMEXT ON SPECTRAL CHANGES IN CATIONIC DYE
Spectral Changes in a Cationic Dye Due: to Interaction with Macromolecules. 11.
Effects of Environment and Macromolecule Structure’
by Robert E. Kay, E. Richard Walwick, and Cheryl K. Gifford Philco Research Laboratories, Newport Beach, California
(Received February 10, 1964)
Studies were conducted to determine the influence of pH, temperature, and dye-niacromolecule ratio on the spectral changes induced by macromolecules in the dye 4,5,4’,5‘dibenzo-3,3’-diethyl-9-methylthiacarbocyanine bromide. Adjustment of the p H to values considerably below the isoionic point prevented interaction of the dye with proteins, whereas a pH above the isoionic point was conducive to the formation of dye-protein complexes. On the other hand, interaction of the dye with DSA was virtually unaffected by pH changes, but important alterations in the wave length of the adsorption iiiaximuiii occurred due to denaturation of the DNA. Dye aggregation induced by inorganic salts was favored by temperatures of less than 20°, and a t higher temperatures the aggregate was dispersed. On the other hand, the aggregation of the dye when adsorbed on a protein was favored by higher temperatures and the interaction of the dye with DNA was not significantly altered by changes in temperature. The stoichiometry of the dye-macromolecule reaction was investigated in the case of DXA and oxidized ribonuclease. The limiting ratio of dye to phosphate groups was 1: 1 for both native and denatured DNA. For oxidized ribonuclease, a ratio of 5.7 : I dye molecules to protein molecules was found. When the ratio was greater than 5.7:1, new peaks formed a t shorter wave lengths. Completely denatured DXA reacted with the dye to produce a n absorption peak a t 535 nip, whereas native DNA induced an absorption peak a t 575 m p . It was found that the ratio of the absorbance a t 535 nip to the absorbance a t 570 nip could serve as a n accurate and sensitive measure of the degree of denaturation. Xative ribonuclease solutions did not interact with the dye to produce new absorption peaks. On the other hand, oxidized ribonuclease reacted with the dye t o form a number of new maxima.
Introduction Previously, a method of detecting macromolecules by the spectral changes in the dye 4,5,4‘,5’-dibenzo3,3’-diethyl-9-methylthiacarbocyaninebromide which result from its interaction with very small amounts of various macromolecules was investigated. From the results obtained, it was concluded that the changes observed were brought about by the adsorption of the dye onto the macromolecular structure. This adsorption process appeared to depend upon the electrostatic forces and it resulted in further polymerization or in depolymerization of the partially polymerized aqueous dye species. Tn macromolecule-dye mixtures, maxima (presumlably representing different degrees of dye polymerization) were found in a t least six distinct
regions of the visible spectrum. The occurrence and exact wave length of each of these maxima depended upon the macromolecule used. Apparently, the structure of the macromolecule and the nature of its anionic sites determined the spectral changes which took place. These findings suggest that environmental factors, through their effects on dye adsorption and macromolecule structure, may alter the degree of dye aggregation which occurs when the dye interacts with a macromolecule. Therefore, we have conducted experiments to : (a) investigate the environmental factors which (1) This research WRS supported by the National Aeronautics and Space Administration under Contract No. NASr-84. (2) R. E. Kay, E. R. Walwick, and C. K . Glfford, J . Phys. Chem., 68, 1896 (1964).
Volume 6 8 , Number 7
July. 1964
R. E. KAY,E. R. WALWICX, ABD C. K. GIFFORD
1908
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