PMID- 8203895
OWN - NLM
STAT- MEDLINE
DCOM- 19940701
LR  - 20131121
IS  - 0003-9861 (Print)
IS  - 0003-9861 (Linking)
VI  - 311
IP  - 2
DP  - 1994 Jun
TI  - Exposure of hydrophobic moieties promotes the selective degradation of hydrogen 
      peroxide-modified hemoglobin by the multicatalytic proteinase complex, 
      proteasome.
PG  - 329-41
AB  - The physiologically relevant stress of a flux of H2O2 increased hemoglobin (Hb) 
      degradation in red blood cells (RBC) and increased the proteolytic susceptibility 
      of Hb in vitro. After exposure to low H2O2 flux rates (6-32 microM/min) Hb 
      exhibited increased exposure of hydrophobic (Trp, Met) and basic (Lys) amino acid 
      R groups, increased hydrophobicity, and increased proteolytic susceptibility 
      during subsequent incubation with RBC extracts, a partially purified preparation 
      called Fraction II (which retains all of the proteolytic activities of RBC 
      extracts), or the purified 670-kDa RBC multicatalytic proteinase complex 
      proteasome. Hydrophobicity was measured by butyl-Sepharose hydrophobic 
      interaction chromatography, by the free energy of transfer from water to ethanol, 
      and by heat denaturation assays. Proteolytic susceptibility was measured by 
      release of free alanine, by fluorescamine-reactive free amino groups, and by 
      release of acid-soluble radioactivity from radiolabeled Hb. Low H2O2 flux rates 
      also caused significant charge changes in Hb (isoelectric focusing gels) and 
      extensive noncovalent aggregation (presumably due to increased hydrophobic 
      interactions) but only limited covalent cross-linking (comparison of sodium 
      dodecyl sulfate-polyacylamide gel electrophoresis (SDS-PAGE) and nondenaturing 
      PAGE). Exposure to higher H2O2 flux rates (56-120 microM/min) caused progressive 
      oxidative destruction of exposed hydrophobic amino acids, decreased 
      hydrophobicity as judged by butyl-Sepharose chromatography and heat denaturation 
      assays, increased hydrophilicity as judged by measurements of the free energy of 
      transfer (delta G') from water to ethanol, and decreased proteolytic 
      susceptibility during incubation with RBC extracts, Fraction II, or purified 
      proteasome. High H2O2 flux rates also caused further charge changes and the 
      extensive formation of covalently cross-linked Hb molecules. Linear regression 
      analyses revealed correlations of 0.8-0.99 for the relationship between Hb 
      hydrophobicity and proteolytic susceptibility for both Fraction II and 
      proteasome. Inhibitor studies and SDS activation experiments indicate that 
      proteasome is responsible for most of the Hb degradation during exposure of RBC 
      to H2O2. Previous work yielded essentially identical conclusions for Hb exposed 
      to hydroxyl radicals (R. E. Pacifici, Y. Kono, and K. J. A. Davies, J. Biol. 
      Chem. 268, 15405-15411, 1993). Thus, nonspecific oxidation by .OH and 
      site-specific (metal-catalyzed) oxidation by H2O2 both yield a more hydrophobic 
      Hb molecule with increased proteolytic susceptibility. We propose that increased 
      exposure of hydrophobic, and perhaps basic, amino acids is the general common 
      cause for degradation of oxidized proteins.(ABSTRACT TRUNCATED AT 400 WORDS)
FAU - Giulivi, C
AU  - Giulivi C
AD  - Institute for Toxicology, University of Southern California, Los Angeles 90033.
FAU - Pacifici, R E
AU  - Pacifici RE
FAU - Davies, K J
AU  - Davies KJ
LA  - eng
GR  - NIEHS E-03598/EH/NCEH CDC HHS/United States
PT  - Journal Article
PT  - Research Support, U.S. Gov't, P.H.S.
PL  - United States
TA  - Arch Biochem Biophys
JT  - Archives of biochemistry and biophysics
JID - 0372430
RN  - 0 (Hemoglobins)
RN  - 0 (Multienzyme Complexes)
RN  - 0 (Oxyhemoglobins)
RN  - BBX060AN9V (Hydrogen Peroxide)
RN  - EC 1.1.3.4 (Glucose Oxidase)
RN  - EC 3.4.22.- (Cysteine Endopeptidases)
RN  - EC 3.4.25.1 (Proteasome Endopeptidase Complex)
SB  - IM
MH  - Animals
MH  - Cattle
MH  - Chromatography, High Pressure Liquid
MH  - Cysteine Endopeptidases/*metabolism
MH  - Electrophoresis, Polyacrylamide Gel
MH  - Erythrocytes/drug effects/*metabolism
MH  - Glucose Oxidase/pharmacology
MH  - Hemoglobins/chemistry/isolation & purification/*metabolism
MH  - Humans
MH  - Hydrogen Peroxide/*pharmacology
MH  - Isoelectric Focusing
MH  - Kinetics
MH  - Multienzyme Complexes/*metabolism
MH  - Oxyhemoglobins/chemistry/isolation & purification/*metabolism
MH  - Proteasome Endopeptidase Complex
MH  - Protein Denaturation
EDAT- 1994/06/01 00:00
MHDA- 1994/06/01 00:01
CRDT- 1994/06/01 00:00
PHST- 1994/06/01 00:00 [pubmed]
PHST- 1994/06/01 00:01 [medline]
PHST- 1994/06/01 00:00 [entrez]
AID - S0003-9861(84)71245-8 [pii]
AID - 10.1006/abbi.1994.1245 [doi]
PST - ppublish
SO  - Arch Biochem Biophys. 1994 Jun;311(2):329-41. doi: 10.1006/abbi.1994.1245.