International Journal of Biological Macromolecules
26 (1999) 135–144
www.elsevier.com/locate/ijbiomac
Role of reducing terminals in unfractionated and low-molecular-mass
heparins in causing free radical generation and loss of structure and
activity of trypsin
Paola Finotti
a,
*, Andrea Pagetta
b
, Carlo Corvaja
c
a
Department of Pharmacology, Uni6ersity of Pado6a, Largo E. Meneghetti
2
,
35131
Pado6a, Italy
b
Department of Organic Chemistry, Uni6ersity of Pado6a, Via F. Marzolo
1
,
35131
Pado6a, Italy
c
Department of Physical Chemistry, Uni6ersity of Pado6a, Via L. Loredan
2
,
35131
Pado6a, Italy
Received 5 January 1999; received in revised form 10 March 1999; accepted 16 March 1999
Abstract
The role of both the length of saccharide chain and reducing terminals in the heparin molecule in causing oxidative effects on
proteins was investigated by employing unfractionated and low-molecular-mass heparins (LMMH), with both intact and reduced
reducing terminals on bovine trypsin. The effects of heparin were found to be dependent on both the concentration and time of
incubation. Heparins with intact reducing terminals caused significantly higher structural and functional alterations of trypsin
compared with heparins with reduced reducing terminals. LMMH was slightly more effective than unfractionated heparin
(UNFH) in reducing structural integrity and inhibiting the amidolytic activity of trypsin when used at the same mass, but not
molar concentrations. Neither the length of saccharide chains nor the number of intact reducing terminals on the heparin molecule
appeared to influence the characteristics of the initial binding of heparin to trypsin, but both these variables crucially affected
linkages which in time mediate the inhibition of catalytic activity and the formation of free radicals, ultimately responsible for
peptide bond cleavage in trypsin. The results suggest that both a critical number of saccharide units, preferentially lying on shorter
chains, and intact reducing terminals in the heparin molecule are involved in setting up the binding which generates radicals and
leads to loss of structure and function of the proteinase. © 1999 Elsevier Science B.V. All rights reserved.
Keywords
:
Trypsin; Heparins; Free radicals; Protein structure
1. Introduction
The effects of heparin on serine proteinases belong-
ing to the coagulation cascade have so far been consid-
ered almost exclusively in terms of acceleration of the
reaction of proteinase inhibition by specific circulating
proteinase inhibitors. This occurs by means of a tem-
plate mechanism, in which the cofactor binds the
proteinase and its inhibitor, tightening the inhibitory
complex [1–3]. While this mechanism is widely recog-
nized to underlie the thrombin–antithrombin reaction
[1] and has also been demonstrated to take place in
other proteinase-inhibitor associations [3], the possibil-
ity that heparin exerts its anticoagulative and an-
tithrombotic properties by acting only on proteinases
has attracted much less attention and has never been
fully elucidated. However, heparin-related anticoagula-
tion involving proteinases, independently of the pres-
Abbre6iations
:
BAPNA, Na-benzoyl-
DL
-arginine p nitroanilide hy-
drochloride; DTNB, 5,5% dithio bis-(2-nitrobenzoic acid); ES-MS,
electrospray mass spectrometry; LMMH, low-molecular-mass hep-
arin; UNFH, unfractionated heparin.
* Corresponding author. Tel.: + 39-049-827-5088; fax: + 39-049-
827-5093; http://www.dfem.unipd.it/.
E-mail address
:
finpa@ux1.unipd.it (P. Finotti)
0141-8130/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved.
PII: S0141-8130(99)00072-0