X-ray microanalysis of toluidine blue stained chromosomes: a quantitative study of the metachromatic reaction of chromatin.
The stoichiometry of metachromatic staining of chromatin by toluidine blue was investigated in isolated metaphase chromosomes from L929 cells using X-ray microanalysis. Microspectrophotometric measurements revealed that a hypsochromic shift (from 595 to 570 nm) occurs in toluidine blue stained chromosomes in relation to the staining solution. Under the electron microscope, stained chromosomes. After toluidine blue staining, X-ray microanalysis of chromosomes revealed a large increase for sulphur counts and a considerable increase for Fe and Cu counts, while the signal of Mg, Ca, Cl, K and Zn was reduced. After subtraction of the intrinsic sulphur signal, S/P ratios of 0.82--for euchromatic arms--and 0.85--for centromeric heterochromatin--were obtained. They are considered representative of dye/DNA phosphate ratios. These results indicate the occurrence of a nearly stoichiometric binding of toluidine blue to chromatin DNA and suggest that an external dye stacking is responsible for the metachromatic staining of metaphase chromosomes.
Stockert
J C
JC
Departmento de Biologia, C-XV, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
Gosálvez
J
J
Del Castillo
P
P
Pelling
C
C
Mezzanotte
R
R
D000818:Animals; D002460:Cell Line; D002843:Chromatin / Q000737:chemistry*; D002875:Chromosomes / Q000648:ultrastructure*; D004577:Electron Probe Microanalysis; D013053:Spectrophotometry; D014048:Tolonium Chloride
D002843:Chromatin; D014048:Tolonium Chloride