Structural analysis of Trypanosoma brucei brucei chromatin by limited proteolysis. An electrical-birefringence study.

The sensitive electric-birefringence method was used to reveal structural differences between the soluble chromatin of procyclic Trypanosoma brucei brucei and the chromatin of the higher eukaryotes. The orientation of the nucleosomal chains and the presence of extended DNA were analysed from the sign and amplitude of the steady-state birefringence, and the conformational properties (overall dimensions and flexibility) were studied in relation to the orientational relaxation times. In contrast to the higher eukaryotes, the birefringence of T.

DNase activity of micrococcal endonuclease covalently immobilized on nylon and polystyrene.

Water-insoluble nucleases were prepared by immobilizing the endonuclease from S. aureus onto the surface of nylon-66 and polystyrene spheres. The activation phase of the synthetic supports was optimized to define optimal conditions of pH, temperature, and Ca2+ concentration for using immobilized enzymes.

Structural differences between the chromatin of procyclic Trypanosoma brucei brucei and of higher eukaryotes as probed by immobilized trypsin.

Soluble chromatin of Trypanosoma brucei brucei procyclic culture forms was submitted to digestion with free or immobilized trypsin. Digestion with trypsin in salt solutions of low and high ionic strengths generated characteristic sets of limit histone peptides. After incubation of chromatin with immobilized trypsin in a solution of low ionic strength, histones were not degraded, whereas a selective proteolysis occurred at 50 mM NaCl.

Enzyme coupling method on calibrated nylon spheres: application to the selective trypsinization of histones in chromatin.

A new method consisting of a two-step activation was developed in order to covalently immobilize enzymes on calibrate nylon 66 spheres. This efficient method associates for the first time peptide bond cleavage and O-alkylation of the support. Optimal conditions for activation and protein coupling were defined, and immobilized trypsin was used to investigate the histone accessibility on chromatin.