char = A;code = 65
char = p;code = 112
char = p;code = 112
char = l;code = 108
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = A;code = 65
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = e;code = 101
char = x;code = 120
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = /;code = 47
char = h;code = 104
char = o;code = 111
char = r;code = 114
char = s;code = 115
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = d;code = 100
char = i;code = 105
char = s;code = 115
char = h;code = 104
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = m;code = 109
char = e;code = 101
char = a;code = 97
char = s;code = 115
char = u;code = 117
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = o;code = 111
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = b;code = 98
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = r;code = 114
char = o;code = 111
char = s;code = 115
char = o;code = 111
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = m;code = 109
char = e;code = 101
char = s;code = 115
char = .;code = 46
result sql = Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes.
Application of the Amplex red/horseradish peroxidase assay to measure hydrogen peroxide generation by recombinant microsomal enzymes.
char = T;code = 84
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = m;code = 109
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = y;code = 121
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = s;code = 115
char = p;code = 112
char = e;code = 101
char = c;code = 99
char = i;code = 105
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = c;code = 99
char = h;code = 104
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = 4;code = 52
char = 5;code = 53
char = 0;code = 48
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = n;code = 110
char = o;code = 111
char = o;code = 111
char = x;code = 120
char = y;code = 121
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = y;code = 121
char = s;code = 115
char = t;code = 116
char = e;code = 101
char = m;code = 109
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = o;code = 111
char = u;code = 117
char = g;code = 103
char = h;code = 104
char = t;code = 116
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = b;code = 98
char = e;code = 101
char = ;code = 32
char = d;code = 100
char = u;code = 117
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = o;code = 111
char = ;code = 32
char = a;code = 97
char = u;code = 117
char = t;code = 116
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = N;code = 78
char = A;code = 65
char = D;code = 68
char = P;code = 80
char = H;code = 72
char = -;code = 45
char = c;code = 99
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = c;code = 99
char = h;code = 104
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = 4;code = 52
char = 5;code = 53
char = 0;code = 48
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = n;code = 110
char = o;code = 111
char = n;code = 110
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = c;code = 99
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = y;code = 121
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = -;code = 45
char = b;code = 98
char = o;code = 111
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = c;code = 99
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = c;code = 99
char = h;code = 104
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = 4;code = 52
char = 5;code = 53
char = 0;code = 48
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = m;code = 109
char = e;code = 101
char = d;code = 100
char = i;code = 105
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = o;code = 111
char = ;code = 32
char = c;code = 99
char = h;code = 104
char = a;code = 97
char = r;code = 114
char = a;code = 97
char = c;code = 99
char = t;code = 116
char = e;code = 101
char = r;code = 114
char = i;code = 105
char = z;code = 122
char = e;code = 101
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = p;code = 112
char = r;code = 114
char = o;code = 111
char = c;code = 99
char = e;code = 101
char = s;code = 115
char = s;code = 115
char = ;code = 32
char = i;code = 105
char = n;code = 110
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = b;code = 98
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = m;code = 109
char = i;code = 105
char = c;code = 99
char = r;code = 114
char = o;code = 111
char = s;code = 115
char = o;code = 111
char = m;code = 109
char = a;code = 97
char = l;code = 108
char = ;code = 32
char = e;code = 101
char = n;code = 110
char = z;code = 122
char = y;code = 121
char = m;code = 109
char = e;code = 101
char = s;code = 115
char = ,;code = 44
char = ;code = 32
char = w;code = 119
char = e;code = 101
char = ;code = 32
char = u;code = 117
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = a;code = 97
char = ;code = 32
char = h;code = 104
char = i;code = 105
char = g;code = 103
char = h;code = 104
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = n;code = 110
char = s;code = 115
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = o;code = 111
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = b;code = 98
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = A;code = 65
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = e;code = 101
char = x;code = 120
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = ;code = 32
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = .;code = 46
char = ;code = 32
char = T;code = 84
char = h;code = 104
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = i;code = 105
char = s;code = 115
char = ;code = 32
char = 2;code = 50
char = 0;code = 48
char = ;code = 32
char = t;code = 116
char = i;code = 105
char = m;code = 109
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = m;code = 109
char = o;code = 111
char = r;code = 114
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = e;code = 101
char = n;code = 110
char = s;code = 115
char = i;code = 105
char = t;code = 116
char = i;code = 105
char = v;code = 118
char = e;code = 101
char = ;code = 32
char = (;code = 40
char = L;code = 76
char = L;code = 76
char = D;code = 68
char = =;code = 61
char = 5;code = 53
char = .;code = 46
char = 0;code = 48
char = p;code = 112
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = /;code = 47
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = L;code = 76
char = L;code = 76
char = Q;code = 81
char = =;code = 61
char = 3;code = 51
char = 0;code = 48
char = p;code = 112
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = /;code = 47
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = y;code = 121
char = );code = 41
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = a;code = 97
char = n;code = 110
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = e;code = 101
char = ;code = 32
char = s;code = 115
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = d;code = 100
char = a;code = 97
char = r;code = 114
char = d;code = 100
char = ;code = 32
char = f;code = 102
char = e;code = 101
char = r;code = 114
char = r;code = 114
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = t;code = 116
char = h;code = 104
char = i;code = 105
char = o;code = 111
char = c;code = 99
char = y;code = 121
char = a;code = 97
char = n;code = 110
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = ;code = 32
char = a;code = 97
char = s;code = 115
char = s;code = 115
char = a;code = 97
char = y;code = 121
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = r;code = 114
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = t;code = 116
char = e;code = 101
char = c;code = 99
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = o;code = 111
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = .;code = 46
char = ;code = 32
char = W;code = 87
char = e;code = 101
char = ;code = 32
char = f;code = 102
char = o;code = 111
char = u;code = 117
char = n;code = 110
char = d;code = 100
char = ;code = 32
char = l;code = 108
char = o;code = 111
char = w;code = 119
char = ,;code = 44
char = ;code = 32
char = b;code = 98
char = u;code = 117
char = t;code = 116
char = ;code = 32
char = d;code = 100
char = e;code = 101
char = t;code = 116
char = e;code = 101
char = c;code = 99
char = t;code = 116
char = a;code = 97
char = b;code = 98
char = l;code = 108
char = e;code = 101
char = ,;code = 44
char = ;code = 32
char = s;code = 115
char = p;code = 112
char = o;code = 111
char = n;code = 110
char = t;code = 116
char = a;code = 97
char = n;code = 110
char = e;code = 101
char = o;code = 111
char = u;code = 117
char = s;code = 115
char = ;code = 32
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = e;code = 101
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = i;code = 105
char = o;code = 111
char = n;code = 110
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = o;code = 111
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = ;code = 32
char = b;code = 98
char = y;code = 121
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = b;code = 98
char = i;code = 105
char = n;code = 110
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = ;code = 32
char = h;code = 104
char = u;code = 117
char = m;code = 109
char = a;code = 97
char = n;code = 110
char = ;code = 32
char = N;code = 78
char = A;code = 65
char = D;code = 68
char = P;code = 80
char = H;code = 72
char = -;code = 45
char = c;code = 99
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = c;code = 99
char = h;code = 104
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = 4;code = 52
char = 5;code = 53
char = 0;code = 48
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = ;code = 32
char = c;code = 99
char = o;code = 111
char = m;code = 109
char = p;code = 112
char = l;code = 108
char = e;code = 101
char = x;code = 120
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = (;code = 40
char = 0;code = 48
char = .;code = 46
char = 0;code = 48
char = 9;code = 57
char = n;code = 110
char = m;code = 109
char = o;code = 111
char = l;code = 108
char = ;code = 32
char = h;code = 104
char = y;code = 121
char = d;code = 100
char = r;code = 114
char = o;code = 111
char = g;code = 103
char = e;code = 101
char = n;code = 110
char = ;code = 32
char = p;code = 112
char = e;code = 101
char = r;code = 114
char = o;code = 111
char = x;code = 120
char = i;code = 105
char = d;code = 100
char = e;code = 101
char = /;code = 47
char = m;code = 109
char = i;code = 105
char = n;code = 110
char = /;code = 47
char = 1;code = 49
char = 0;code = 48
char = 0;code = 48
char = U;code = 85
char = n;code = 110
char = i;code = 105
char = t;code = 116
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
char = N;code = 78
char = A;code = 65
char = D;code = 68
char = P;code = 80
char = H;code = 72
char = -;code = 45
char = c;code = 99
char = y;code = 121
char = t;code = 116
char = o;code = 111
char = c;code = 99
char = h;code = 104
char = r;code = 114
char = o;code = 111
char = m;code = 109
char = e;code = 101
char = ;code = 32
char = P;code = 80
char = 4;code = 52
char = 5;code = 53
char = 0;code = 48
char = ;code = 32
char = r;code = 114
char = e;code = 101
char = d;code = 100
char = u;code = 117
char = c;code = 99
char = t;code = 116
char = a;code = 97
char = s;code = 115
char = e;code = 101
char = );code = 41
char = .;code = 46
char = ;code = 32
char = S;code = 83
char = i;code = 105
char = g;code = 103
char = n;code = 110
char = i;code = 105
char = f;code = 102
char = i;code = 105
char = c;code = 99
char = a;code = 97
char = n;code = 110
char = t;code = 116
char = l;code = 108
char = y;code = 121
char = ;code = 32
char = h;code = 104
char = i;code = 105
char = g;code = 103
char = h;code = 104
char = e;code = 101
char = r;code = 114
char = ;code = 32
char = r;code = 114
char = a;code = 97
char = t;code = 116
char = e;code = 101
char = s;code = 115
char = ;code = 32
char = o;code = 111
char = f;code = 102
char = ;code = 32
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result sql = The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.

Publication: Free Radic. Biol. Med. 2010 Jun 1 ;48(11)::1485-91. :doi: 10.1016/j.freeradbiomed.2010.02.030.
Publication Date: 2010 Jun 1
Study Author(s): Mishin, Vladimir;Gray, Joshua P;Heck, Diane E;Laskin, Debra L;Laskin, Jeffrey D;
Institution: Pharmacology and Toxicology, Rutgers University, Piscataway, NJ 08854, USA.
Shortcut link to this study:
The formation of reactive oxygen species by the cytochrome P450 monooxygenase system is thought to be due to autoxidation of NADPH-cytochrome P450 reductase and the nonproductive decay of oxygen-bound cytochrome P450 intermediates. To characterize this process in recombinant microsomal enzymes, we used a highly sensitive hydrogen peroxide assay based on Amplex red oxidation. This assay is 20 times more sensitive (LLD=5.0pmol/assay and LLQ=30pmol/assay) than the standard ferrous thiocyanate assay for detection of hydrogen peroxide. We found low, but detectable, spontaneous generation of hydrogen peroxide by recombinant human NADPH-cytochrome P450 reductase complexes (0.09nmol hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). Significantly higher rates of hydrogen peroxide production were observed when recombinant cytochrome P450 enzymes were coexpressed with NADPH-cytochrome P450 reductase (0.31nmol of hydrogen peroxide/min/100Units of NADPH-cytochrome P450 reductase). This was independent of the addition of any exogenous cytochrome P450 substrates. These data demonstrate that cytochrome P450s are a major source of hydrogen peroxide in the recombinant cytochrome P450 monooxygenase system. Moreover, substrate binding is not required for the cytochrome P450s to generate reactive oxygen species.
PMID: 20188819

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