TY - JOUR
T1 - TRANSIENT EFFECTS in the ACRYLAMIDE QUENCHING of SINGLE TRYPTOPHAN PROTEINS, OBSERVED USING FREQUENCY-DOMAIN FLUOROMETRY
AU - Lakowicz, Joseph R.
AU - Johnson, Michael L.
AU - Gryczynski, Ignacy
AU - Szmacinski, Henryk
AU - Joshi, N.
AU - Laczko, G.
PY - 1988/6/24
Y1 - 1988/6/24
N2 - We used GHz frequency-domain fluorometry to examine the tryptophan intensity decays of NATA (N-acetyl-L-tryptophanamide), gly-trp-gly, and the single tryptophan proteins ACTH, S. nuclease and ribonuclease T, (RNase T,). In all cases the intensity decays became more heterogeneous in the presence of quenching, which we attribute to a time-dependent rate constant for quenching (transient effects). The frequency-domain data were analyzed using the Smoluchowski model (exp(-T /t-2b / and the radiation boundary condition (RBC) model. In contrast to the 1-17 model, the RBC model does not assume the fluorophore-quencher pair is immediately deactivated, but rather assumes a rate constant for deactivation of the pair (K) as well as a mutual diffusion coefficient (D). The RBC model provides dramatically improved fits to the data. The values of both D and decreases progressively in the order listed above, which is with decreasing exposure to the aqueous phase. Because the RBC model may not be strictly correct in homogeneous solution, and probably less so in the hindered anisotropic environment of the proteins, the recovered values of D and K should be regarded as apparent values. The recovered intensity decays can be compared with molecular dynamic calculations of quencher trajectories in proteins.
AB - We used GHz frequency-domain fluorometry to examine the tryptophan intensity decays of NATA (N-acetyl-L-tryptophanamide), gly-trp-gly, and the single tryptophan proteins ACTH, S. nuclease and ribonuclease T, (RNase T,). In all cases the intensity decays became more heterogeneous in the presence of quenching, which we attribute to a time-dependent rate constant for quenching (transient effects). The frequency-domain data were analyzed using the Smoluchowski model (exp(-T /t-2b / and the radiation boundary condition (RBC) model. In contrast to the 1-17 model, the RBC model does not assume the fluorophore-quencher pair is immediately deactivated, but rather assumes a rate constant for deactivation of the pair (K) as well as a mutual diffusion coefficient (D). The RBC model provides dramatically improved fits to the data. The values of both D and decreases progressively in the order listed above, which is with decreasing exposure to the aqueous phase. Because the RBC model may not be strictly correct in homogeneous solution, and probably less so in the hindered anisotropic environment of the proteins, the recovered values of D and K should be regarded as apparent values. The recovered intensity decays can be compared with molecular dynamic calculations of quencher trajectories in proteins.
UR - http://www.scopus.com/inward/record.url?scp=84935176056&partnerID=8YFLogxK
U2 - 10.1117/12.945383
DO - 10.1117/12.945383
M3 - Conference article
AN - SCOPUS:84935176056
SN - 0277-786X
VL - 909
SP - 170
EP - 177
JO - Proceedings of SPIE - The International Society for Optical Engineering
JF - Proceedings of SPIE - The International Society for Optical Engineering
T2 - Time-Resolved Laser Spectroscopy in Biochemistry 1988
Y2 - 11 January 1988 through 17 January 1988
ER -