Avtor/Urednik		Žerovnik, Eva; Kopitar, Gregor; Kos, Janko; Turk, Vito
Naslov		Refolding of recombinant sulphonated procathepsin S and of reduced chicken cystatin; implications for renaturation experiments
Tip		članek
Vir		Biochim Biophys Acta
Vol. in št.		Letnik 1383
Leto izdaje		1998
Obseg		str. 211-8
Jezik		eng
Abstrakt		Kinetic stopped-flow measurements of refolding of the recombinant sulphonated procathepsin S from 6 M urea are presented. The experiments were performed using intrinsic tryptophan fluorescence and fluorescence of the hydrophobic probe 1-anilino-naphthalene-8-sulfonate (ANS). Initially, (t 1/ 2 = 3 +- 1 ms) an intermediate with increased ANS fluorescence and protected tryptophan environment is formed. Much later, a slow increase in ANS fluorescence occurs with no accompanying changes in tryptophan fluorescence. The reaction of the slow ANS fluorescence increase correlates with the rate of aggregation as shown by the size exclusion chromatography (SEC). For comparison, the folding reactions of the reduced chicken cystatin were measured, both, by intrinsic tryptophan and extrinsic ANS fluorescence. An early intermediate forms very fast in the refolding of reduced chicken cystatin on 6-fold dilution from 5.7 M GuHCl (t1/2 = 5 +-2 ms), similarly to that observed for the sulphonated procathepsin S. ANS fluorescence and tryptophan fluorescence decrease further (t1/2 =100+- 50 ms) leading to a late, 'more structured' intermediate which is prone to dimerization. (C) 1998 Elsevier Science B.V.
Deskriptorji		CATHEPSINS CYSTATINS PROTEIN FOLDING CHICKENS RECOMBINANT PROTEINS