EXPOSURE OF HYDROPHOBIC MOIETIES PROMOTES THE SELECTIVE DEGRADATION OF HYDROGEN PEROXIDE-MODIFIED HEMOGLOBIN BY THE MULTICATALYTIC PROTEINASE COMPLEX, PROTEASOME
C. Giulivi et al., EXPOSURE OF HYDROPHOBIC MOIETIES PROMOTES THE SELECTIVE DEGRADATION OF HYDROGEN PEROXIDE-MODIFIED HEMOGLOBIN BY THE MULTICATALYTIC PROTEINASE COMPLEX, PROTEASOME, Archives of biochemistry and biophysics, 311(2), 1994, pp. 329-341
The physiologically relevant stress of a flux of H2O2 increased hemogl
obin (Hb) degradation in red blood cells (REC) and increased the prote
olytic susceptibility of Hb in vitro. After exposure to low H2O2 flux
rates (6-32 mu M/ min) Hb exhibited increased exposure of hydrophobic
(Trp, Met) and basic (Lys) amino acid R groups, increased hydrophobici
ty, and increased proteolytic susceptibility during subsequent incubat
ion with RBC extracts, a partially purified preparation called Fractio
n II (which retains all of the proteolytic activities of RBC extracts)
, or the purified 670-kDa RBC multicatalytic proteinase complex protea
some. Hydrophobicity was measured by butyl-Sepharose hydrophobic inter
action chromatography, by the free energy of transfer from water to et
hanol, and by heat denaturation assays. Proteolytic susceptibility was
measured by release of free alanine, by fluorescamine-reactive free a
mino groups, and by release of acid-soluble radioactivity from radiola
beled Hb. Low H2O2 flux rates also caused significant charge changes i
n Rb (isoelectric focusing gels) and extensive noncovalent aggregation
(presumably due to increased hydrophobic interactions) but only limit
ed covalent cross-linking (comparison of sodium dodecyl suIfate-polyac
ylamide gel electrophoresis (SDS-PAGE) and nondenaturing PAGE). Exposu
re to higher H2O2 flux rates (56-120 mu M/min) caused progressive oxid
ative destruction of exposed hydrophobic amino acids, decreased hydrop
hobicity as judged by butyl-Sepharose chromatography and heat denatura
tion assays, increased hydrophilicity as judged by measurements of the
free energy of transfer (Delta G') from water to ethanol, and decreas
ed proteolytic susceptibility during incubation with RBC extracts, Fra
ction II, or purified proteasome. High H2O2 flux rates also caused fur
ther charge changes and the extensive formation of covalently cross-li
nked Hb molecules. Linear regression analyses revealed correlations of
0.8-0.99 for the relationship between Hb hydrophobicity and proteolyt
ic susceptibility for both Fraction II and proteasome. Inhibitor studi
es and SDS activation experiments indicate that proteasome is responsi
ble for most of the Hb degradation during exposure of RBC to H2O2. Pre
vious work yielded essentially identical conclusions for Hb exposed to
hydroxyl radicals (R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. B
iol. Chem. 268, 15405-15411, 1993). Thus, nonspecific oxidation by OH
and site-specific (metal-catalyzed) oxidation by H2O2 both yield a mor
e hydrophobic Hb molecule with increased proteolytic susceptibility. W
e propose that increased exposure of hydrophobic, and perhaps basic, a
mino acids is the general common cause for degradation of oxidized pro
teins. Since peptide bonds flanked by hydrophobic or basic amino acids
are the preferred substrates for proteasome, our results indicate tha
t partial protein unfolding is the key to increased proteolysis during
oxidative stress. Covalently cross-linked protein aggregates, generat
ed during extreme oxidative stress conditions, are suggested to be poo
r substrates for proteolysis due to stearic hinderance of their intera
ction with proteasome. (C) 1994 Academic Press, Inc.