Purification of five bovine luteinizing hormone (bLH) isoforms. Chemical physical, biological and inmunological characterization

Abstract

THE STUDY DESCRIBES THE PURIFICATION OF FIVE LH ISOFORMS FROM BOVINE PITUITARY EXTRACTS. CATIONIC INTERCHANGE CHROMATOGRAPHY WAS USED TO OBTAIN FRACTIONS AT A PH OF 6.8 AND 9.5. THESE WERE THEN REPURIFIED UNDER IDENTICAL CONDITIONS IN DEAE-SEPHACEL TO OBTAIN LH-I (1.2 MG/KG OF PITUITARY), AND LH-II (1.8 MG/KG), LH-III (1.5 MG/KG), LH-IV (1.2 MG/KG) AND LH-V (7.0 MG/KG), RESPECTIVELY. THE RELATIVE MOBILITY (RF) OF EACH ISOFORM WAS DETERMINED USING POLYACRYLAMIDE GEL ELECTROPHORESIS UNDER NATIVE CONDITIONS. LH-I PRESENTED AN RF OF 0.16 CM; LH- II, LH-III AND LH-IV HAD AN RF OF 0.044 CM, WHILE LHV HAD A 0.11 CM RF. THE MOLECULAR WEIGHT (MW) OF LH-I AND LH-V WAS 37.5 KDA, WHILE LH-II, LH-III AND LH-IV HAD A MW OF 38.5 KDA. THE IMMUNOTRANSFERENCE ANALYSIS IDENTIFIED MW BANDS WEIGHING 59.0, 37.5 AND 22.0 KDA IN THE STANDARDS AS WELL AS IN THE DIFFERENT ISOFORMS. THE IN VIVO BIOLOGICAL POTENCY CORRESPONDED TO 1.04 U/MG (LH-I); 5.9 U/MG (LH-II); 0.16 U/MG (LH-III); 0.86 U/MG (LH-IV) AND 1.4 U/MG (LH-V) AND THEIR LH RADIOINMMUNOASSAY (RIA) 50% EXPECTED DOSE (ED50) WERE 2.27 NG/TUBE (LH-I), 1.19 NG/TUBE (LH-II), 1.35 NG/TUBE (LH-III), 0.66 NG/TUBE (LH-IV) AND 1.71 NG/TUBE (LH-V). THESE DATA SHOW THAT IT IS POSSIBLE TO OBTAIN TWO LH ISOFORMS AT PH 6.8 (I AND V) AND THREE AT PH 9.5 (II, III AND IV), WITH HETEROGENEOUS CHARGES, RIA AND BIOLOGICAL ACTIVITY, WITH SUFFICIENT YIELD AS TO TRY TO DEVELOP SIMPLER AND MORE PRECISE ANALYTICAL METHODS.