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Cyclic voltammetry and UV/VIS spectrometry studies show that 4'-(9-acridinylamino)-methanesulfon- m -anisidide (mAMSA) can be oxidized electrochemically to N 1 -methylsulfonyl- N 4 -(9-acridinyl)-3-methoxy-2, 5-cyclohexadiene-1,4-diimine (mAQDI) in Tris buffer, pH 7.5. The formal potential of this 2-electron process, as determined by spectroelectrochemical techniques, was 0.141 V versus saturated calomel electrode. Voltammetric data also indicate that an electron transfer reaction between mAMSA and Cu(II) was thermodynamically favored. Two lines of evidence suggest that mAQDI and Cu(I) are the active species in DNA breakage: (1) mAQDI, in the presence of Cu(I), induced both single- and double-strand DNA breakage of the superhelical pDPT275 form I DNA. mAQDI or Cu(I), when used alone, was less effective. (2) The DNA-breaking activity of an mAMSA-Cu(II) mixture was kinetically correlated with the production of both Cu(I) and mAQDI. Thin-layer Chromatographie studies showed that mAMSA was oxidized to mAQDI which, in turn, was hydrolyzed. The end product was identified as 9-aminoacridine. When DNA breakage activity was measured as a function of reaction time, a biphasic response was observed. Maximal DNA-breaking activity was obtained upon mixing mAMSA and Cu(II) for 2–4 hr, depending on the concentrations of mAMSA and Cu(II), and was followed by a subsequent decrease in breakage. The decrease appears to be due to the decrease in Cu(I) production and the hydrolysis of mAQDI. These results substantiate the proposed mechanism that DNA breakage induced by mAMSA-Cu(II) involves a rate-limiting electron transfer step to form mAQDI and Cu(I), which are the active species for DNA breakages.

4′-(9-Acridinylamino)methanesulfon- m -anisidide (mAMSA) interacts with red cell membranes, resulting in the formation of fluorescent protein adducts. The mAMSA-membrane protein adducts exhibited an emission fluorescence maximum at 445 nm, with two shoulders at approximately 425 and 470 nm. The major labeled proteins were identified as spectrins 1 and 2 and bands 3, 4.1, 4.2 and 5. The fluorescence intensity increased with increasing mAMSA concentrations (0.03 to 1.5 mM), time (15–120 min), and temperature of the reaction. Results from sodium dodecyl sulfate gel electrophoresis show that mAMSA caused no detectable change in the molecular weight of membrane proteins. This indicates that mAMSA is a monofunctional, noncrosslinking agent. Other acridine analogs, 9-aminoacridine and acridine, did not fluorescently label membrane proteins, suggesting that the presence of the acridine nucleus is not sufficient for labeling. Addition of 2-mercaptoethanol to the mAMSA-membrane reaction mixtures reversed the fluorescence labeling. Furthermore, pretreatment of membrane proteins with N -ethylmaleimide or iodoacetamide prevented the formation of fluorescent mAMSA-membrane protein adducts. These data suggest that mAMSA interacts with sulfhydryl groups of the membrane proteins. When the membrane sulfhydryl groups were assayed by labeling with N -[ ethyl -2- 3 H ]ethylmaleimide, it was shown that the accessible membrane sulfhydryl groups were reduced after the mAMSA treatment. The above results suggest that mAMSA covalently binds to the sulfhydryl groups in the red cell membrane, with the production of fluorescent mAMSA-protein adducts.

We investigated the interactions of 4′-(9-acridinylamino)methanesulfon- m -anisidide (mAMSA) with thiol-containing compounds and the potential binding of the thiolytic adducts to DNA. All thiols tested (glutathione, cysteine, coenzyme A, 2-mercaptoethanol and lactate dehydrogenase) formed adducts with mAMSA as evidenced by changes in the absorption spectrum of mAMSA and induction of fluorescence. Spectral changes induced by the thiols were different, suggesting that each thiol induced specific changes in the electronic structure of the acridine nucleus. Treatment of glutathione with p -chloromercuribenzoate eliminated the absorption spectral changes and induction of fluorescence, indicating that the reduced-thiol group is involved. In high ionic strength buffer, addition of calf thymus DNA induced fluorescence-quenching of both the mAMSA-glutathione and mAMSA-cysteine adducts without spectral shift. Viscometric studies showed that mAMSA and mAMSA-glutathione intercalated into DNA and produced similar increases in the length of linear DNA.