A rapid, sensitive and economical assessment of monoclonal antibody conformational stability by intrinsic tryptophan fluorescence spectroscopy

2008 ◽  
Vol 3 (9-10) ◽  
pp. 1201-1211 ◽  
Author(s):  
Patrick Garidel ◽  
Matthias Hegyi ◽  
Stefan Bassarab ◽  
Michael Weichel
Keyword(s):  
Monoclonal Antibody ◽  
Fluorescence Spectroscopy ◽  
Conformational Stability ◽  
Tryptophan Fluorescence ◽  
Intrinsic Tryptophan Fluorescence

scholarly journals Intrinsic tryptophan fluorescence spectroscopy reliably determines galectin-ligand interactions

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Paulina Sindrewicz ◽  
Xiaoxin Li ◽  
Edwin A. Yates ◽  
Jeremy E. Turnbull ◽  
Lu-Yun Lian ◽  
...  
Keyword(s):  
Fluorescence Spectroscopy ◽  
Tryptophan Fluorescence ◽  
Intrinsic Tryptophan Fluorescence ◽  
Ligand Interactions

scholarly journals Serpin α1proteinase inhibitor probed by intrinsic tryptophan fluorescence spectroscopy

1996 ◽  
Vol 5 (11) ◽  
pp. 2226-2235 ◽  
Author(s):  
Henryk Koloczek ◽  
Agnieszka Banbula ◽  
Jan Potempa ◽  
Guy S. Salvesen
Keyword(s):  
Fluorescence Spectroscopy ◽  
Tryptophan Fluorescence ◽  
Intrinsic Tryptophan Fluorescence

scholarly journals Inactivation of sarcoplasmic-reticulum Ca2+-ATPase in low-frequency-stimulated muscle results from a modification of the active site

1992 ◽  
Vol 285 (1) ◽  
pp. 303-309 ◽  
Author(s):  
S Matsushita ◽  
D Pette
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Active Site ◽  
Low Frequency ◽  
Fluorescein Isothiocyanate ◽  
Tryptophan Fluorescence ◽  
Atp Binding ◽  
Binding Characteristics ◽  
Intrinsic Tryptophan Fluorescence ◽  
Inactive Form ◽  
Induced Changes

Molecular changes underlying the partial inactivation of the sarcoplasmic-reticulum (SR) Ca(2+-) ATPase in low-frequency-stimulated fast-twitch muscle were investigated in the present study. The specific Ca(2+)-ATPase activity, as well as the ATP- and acetyl phosphate-driven Ca2+ uptakes by the SR, were reduced by approx. 30% in 4-day-stimulated muscle. Phosphoprotein formation of the enzyme in the presence of ATP or Pi was also decreased to the same extent. Measurements of ATP binding revealed a 30% decrease in binding to the enzyme. These changes were accompanied by similar decreases in the ligand-induced (ATP, ADP, Pi) intrinsic tryptophan fluorescence. A decreased binding of fluorescein isothiocyanate (FITC) corresponded to the lower ATP binding and phosphorylation of the enzyme. Moreover, Pi-induced changes in fluorescence of the FITC-labelled enzyme did not differ between SR from stimulated and contralateral muscles, indicating that Ca(2+)- ATPase molecules which did not bind FITC were responsible for the decreased Pi-dependent phosphorylation, and therefore represented the inactive form of the enzyme. No differences existed between the Ca(2+)-induced changes in the intrinsic fluorescence of SR from stimulated and contralateral muscles which fit their similar Ca(2+)-binding characteristics. Taking the proposed architecture of the Ca2(+)-ATPase into consideration, our results suggest that the inactivation relates to a circumscribed structural alteration of the enzyme in sections of the active site consisting of the nucleotide-binding and phosphorylation domains.

scholarly journals Substrate-induced conformational transition in human phenylalanine hydroxylase as studied by surface plasmon resonance analyses: the effect of terminal deletions, substrate analogues and phosphorylation

2003 ◽  
Vol 369 (3) ◽  
pp. 509-518 ◽  
Author(s):  
Anne J. STOKKA ◽  
Torgeir FLATMARK
Keyword(s):  
Surface Plasmon Resonance ◽  
Surface Plasmon ◽  
Plasmon Resonance ◽  
Phenylalanine Hydroxylase ◽  
Conformational Transition ◽  
Tryptophan Fluorescence ◽  
Catalytic Core ◽  
Intrinsic Tryptophan Fluorescence ◽  
Human Phenylalanine Hydroxylase ◽  
Activity State

The optical biosensor technique, based on the surface plasmon resonance (SPR) phenomenon, was used for real-time measurements of the slow conformational transition (isomerization) which occurs in human phenylalanine hydroxylase (hPAH) on the binding/dissociation of l-phenylalanine (l-Phe). The binding to immobilized tetrameric wt-hPAH resulted in a time-dependent increase in the refractive index (up to approx. 3min at 25°C) with an end point of approx. 75RU (resonance units)/(pmolsubunit/mm2). By contrast, the contribution of binding the substrate (165Da) to its catalytic core enzyme [ΔN(1—102)/ΔC(428—452)-hPAH] was only approx. 2RU/(pmolsubunit/mm2). The binding isotherm for tetrameric and dimeric wt-hPAH revealed a [S]0.5-value of 98±7μM (h = 1.0) and 158±11μM, respectively, i.e. for the tetramer it is slightly lower than the value (145±5μM) obtained for the co-operative binding (h = 1.6±0.4) of l-Phe as measured by the change in intrinsic tryptophan fluorescence. The responses obtained by SPR and intrinsic tryptophan fluorescence are both considered to be related to the slow reversible conformational transition which occurs in the enzyme upon l-Phe binding, i.e. by the transition from a low-activity state ('T-state') to a relaxed high-activity state ('R-state') characteristic of this hysteretic enzyme, however, the two methods reflect different elements of the transition. Studies on the N- and C-terminal truncated forms revealed that the N-terminal regulatory domain (residues 1—117) plus catalytic domain (residues 118—411) were required for the full signal amplitude of the SPR response. Both the on- and off-rates for the conformational transition were biphasic, which is interpreted in terms of a difference in the energy barrier and the rate by which the two domains (catalytic and regulatory) undergo a conformational change. The substrate analogue 3-(2-thienyl)-l-alanine revealed an SPR response comparable with that of l-Phe on binding to wild-type hPAH.

scholarly journals Interaction of caldesmon with phospholipids

1993 ◽  
Vol 291 (2) ◽  
pp. 403-408 ◽  
Author(s):  
E A Czuryło ◽  
J Zborowski ◽  
R Dabrowska
Keyword(s):  
Fluorescence Spectroscopy ◽  
Hydrophobic Interactions ◽  
Tryptophan Fluorescence ◽  
Terminal Part ◽  
Strong Binding ◽  
Terminal Fragment ◽  
Electrostatic And Hydrophobic Interactions ◽  
The Stability

The interaction of caldesmon with liposomes composed of various phospholipids has been examined by tryptophan fluorescence spectroscopy. The results indicate that caldesmon makes its strongest complex with phosphatidylserine (PS) vesicles (Kass. = 1.45 x 10(5) M-1). Both electrostatic and hydrophobic interactions contribute to the stability of this complex. The site for strong binding of PS seems to be located in the N-terminal part of the 34 kDa C-terminal fragment of caldesmon. Binding of PS at this site results in displacement of calmodulin from its complex with caldesmon.

scholarly journals An intrinsic-tryptophan-fluorescence study of phage phi29 connector/nucleic acid interactions

1994 ◽  
Vol 225 (2) ◽  
pp. 747-753 ◽  
Author(s):  
Maria Angeles Urbaneja ◽  
Susana Rivas ◽  
Jose L. Carrascosa ◽  
Jose Maria Valpuesta
Keyword(s):  
Nucleic Acid ◽  
Tryptophan Fluorescence ◽  
Fluorescence Study ◽  
Intrinsic Tryptophan Fluorescence ◽  
Nucleic Acid Interactions
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