Subject(s)
Ethanol/analogs & derivatives , Immunoglobulin Fc Fragments/metabolism , Immunoglobulin G/metabolism , Nitrobenzenes , Staphylococcal Protein A/metabolism , Animals , Electrophoresis, Polyacrylamide Gel , Immunoglobulin Fc Fragments/radiation effects , Immunoglobulin G/radiation effects , Mice , Protein Binding , Spectrophotometry , Staphylococcal Protein A/radiation effects , Ultraviolet RaysABSTRACT
Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.
Subject(s)
Lasers , Proteins/antagonists & inhibitors , Proteins/radiation effects , Acetylcholinesterase/radiation effects , Alkaline Phosphatase/antagonists & inhibitors , Animals , Antibodies , Biophysical Phenomena , Biophysics , Humans , In Vitro Techniques , Proteins/immunology , Rosaniline Dyes , Serum Albumin, Bovine/antagonists & inhibitors , Staphylococcal Protein A/radiation effects , beta-Galactosidase/antagonists & inhibitorsABSTRACT
Several proteins have been crosslinked to DNA by low dose uv irradiation. The principle of the method is based on an efficient and fast radiation induced reaction of amino acid residues with DNA at low pH. The method seems to be of general applicability for crosslinking proteins to DNA in a very simple one step procedure. Some of such DNA-protein conjugates have been used as probes for hybridization experiments. DNA-protein A probes were found to be most useful.
