International Journal of Pharmaceutics 294 (2005) 193–199
Second derivative tryptophan ﬂuorescence spectroscopy as a tool
to characterize partially unfolded intermediates of proteins
, Vikas K. Sharma
, Devendra S. Kalonia
Department of Pharmaceutical Sciences, School of Pharmacy, University of Connecticut, U-2092 Storrs, CT 06269, USA
Institute of Materials Science, University of Connecticut, Storrs, CT 06269, USA
Received 28 October 2004; received in revised form 21 January 2005; accepted 26 January 2005
The application of second derivative tryptophan (Trp) ﬂuorescence spectroscopy to characterize partially unfolded interme-
diates of proteins relevant to protein formulation was investigated. The second derivatives of the normalized emission scans of
N-acetyl tryptophanamide (NATA), single-Trp containing proteins, somatostatin and human serum albumin (HSA), and two-
Trp containing proteins previously shown to form partially unfolded intermediates, ␤-lactoglobulin (␤Lg) and interferon ␣-2a
(IFN␣2a), were studied in solution. The second derivative of NATA in water showed three bands at 340, 348 and 367 nm. The
340nm band showed a blue shift, whereas the intensity of all three bands was affected by a decrease in solution polarity. Second
derivative of single-Trp containing proteins, somatostatin and HSA, showed three negative bands, whereas, the second derivative
of the two-Trp containing proteins, ␤Lg and IFN␣2a, showed four bands, two of which lie in the 320–340nm range. These two
bands were attributed to the presence of the Trps in different microenvironments. The characteristic changes in the intensities
of these two bands on addition of guanidine hydrochloride (␤Lg) and with a decrease in solution pH (IFN␣2a) were related to
the presence of partially unfolded intermediates of these proteins. Thus, second derivative Trp ﬂuorescence spectroscopy can be
used as an important tool to identify partially unfolded states of proteins during formulation utilizing order of magnitude lower
concentrations compared to such other technique as near UV CD.
© 2005 Elsevier B.V. All rights reserved.
Keywords: Second derivative; Fluorescence; Tryptophan; Proteins; Tertiary structure; Partially unfolded intermediates
Corresponding author. Tel.: +1 860 486 3655;
fax: +1 860 486 4998.
E-mail address: firstname.lastname@example.org (D.S. Kalonia).
Department of Chemistry, Massachusetts Institute of Technology,
Cambridge, MA 02139, USA.
Protein conformation plays an important role in un-
derstanding the physical stability (unfolding, aggre-
gation and precipitation) of proteins (Manning et al.,
1989; Arakawa et al., 1993; Wang, 1999). Certain pro-
teins can form stable partially unfolded intermediates,
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