Selective photochemical modification by trichloroethanol of tryptophan residues in proteins with a high tyrosine-to-tryptophan ratio.
We present an improved procedure for the selective modification of tryptophan residues in proteins. A simple, low-cost set-up allows rapid tryptophan photoreaction upon ultraviolet irradiation in the presence of 2,2,2-trichloroethanol. This photochemical reaction is carried out under native conditions, occurs only in the excited state of tryptophan, and yields a single, as yet unidentified, photoproduct. Except for tyrosine, no reaction with other amino acid side chains are known. Stringent photoselection of tryptophan, ensuring that tyrosine residues are not affected, is achieved in situ without the need for an elaborate system of optical filters or lenses. Illumination with a medium-wave uv lamp of samples placed in disposable, dual pathlength, polystyrene fluorescence cuvettes allows treatment of small sample volumes (greater than or equal to 100 microliters) of various optical density. Chromophore accessibility in oligomeric assemblies or protein-nucleic acid complexes can be assessed by this reaction since the integrity of these structures is preserved. Moreover, this technique can be used to evaluate the involvement of tryptophan residues in catalytic or ligand binding processes.
Casas-Finet
J R
JR
Department of Chemistry and Biochemistry, University of Maryland Baltimore County 21228.
Wilson
S H
SH
Karpel
R L
RL
D000818:Animals; D002417:Cattle; D004265:DNA Helicases / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D005023:Ethylene Chlorohydrin / Q000031:analogs & derivatives* / Q000737:chemistry; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D018398:Homeodomain Proteins; D015778:Minor Histocompatibility Antigens; D010777:Photochemistry; D019253:Proto-Oncogene Proteins c-bcl-2; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry; D051818:Replication Protein C; D012097:Repressor Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013951:Thymus Hormones / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry*; D014443:Tyrosine / Q000737:chemistry*
C096816:BCL2-related protein A1; D004268:DNA-Binding Proteins; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D019253:Proto-Oncogene Proteins c-bcl-2; D011994:Recombinant Proteins; D012097:Repressor Proteins; D012261:Ribonucleoproteins; D029701:Saccharomyces cerevisiae Proteins; D013951:Thymus Hormones; D014443:Tyrosine; D005023:Ethylene Chlorohydrin; D014364:Tryptophan; C005849:2,2,2-trichloroethanol; D004265:DNA Helicases; D051818:Replication Protein C