Specific assay of serum lactate dehydrogenase isoenzyme 1 by proteolysis with alpha-chymotrypsin and protein denaturation.
We devised a method for assaying serum lactate dehydrogenase isoenzyme 1 (LD-1) activity specifically by preincubation with alpha-chymotrypsin and guanidine. Cleavage of phenylalanine bonds in the loop of A and B subunits of LD-3, LD-4, and LD-5 isoenzymes (residues 117-119) by incubation with alpha-chymotrypsin for a short time completely inactivated these isoenzymes and partially inactivated LD-2. Addition of guanidine (0.50 mol/L, pH 7.8) to the incubation mixture containing the chymotrypsin completed the inactivation of LD-2. As much as 4000 U/L of LD-2, LD-3, LD-4, and LD-5 were inactivated, whereas LD-1 was affected only slightly. Results by this method (y) correlated well with those by the Roche Isomune immunochemical LD-1 method (x): y = 0.98 x -0.11, r = 0.99 (n = 60). Within-run CVs were 0.5-2.5%. Several common interferents had no effect. In 500 healthy people, serum LD-1 ranged between 66 and 130 U/L, with a mean +/- SD of 88 +/- 15 U/L.
Shirahase
Y
Y
Department of Research and Development, International Reagent Corp., Kobe, Japan.
Watazu
Y
Y
Kaneda
N
N
Uji
Y
Y
Okabe
H
H
Karmen
A
A
D002918:Chymotrypsin / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D019791:Guanidine; D006146:Guanidines / Q000378:metabolism; D006801:Humans; D007118:Immunoassay / Q000379:methods / Q000706:statistics & numerical data; D007527:Isoenzymes; D007770:L-Lactate Dehydrogenase / Q000037:antagonists & inhibitors / Q000097:blood*; D010649:Phenylalanine / Q000378:metabolism; D011489:Protein Denaturation; D012016:Reference Values
D006146:Guanidines; D007527:Isoenzymes; D010649:Phenylalanine; D007770:L-Lactate Dehydrogenase; D002918:Chymotrypsin; D019791:Guanidine