Spectroscopic analysis of the stability of bothrops myotoxic phospholipases A2 to guanidine and urea denaturation.
Spectrophotometric profiles representing the unfolding induced by guanidine on Bothrops moojeni myotoxins-I (MjTX-I) and II (MjTX-II), Bothrops jararacussu bothropstoxin-I (BthTX-I) and Bothrops pirajai piratoxin-I (PrTX-I) were obtained and compared with those obtained with bovine ribonuclease A (RNAse) and trypsin. The molar (epsilon(1M)) and percent (epsilon(1%)) extinction coefficients were determined for the four myotoxins as well as for RNAse and trypsin as reference parameters. These coefficients were then used throughout this work. The changes in free energy (deltaGD(H)(2)(O)) corresponding to zero guanidine concentration and the guanidine concentrations (D(1/2)) able to convert 50% of the molecules from the native to the unfolded state were determined. The values of deltaGD (H)(2)(O) ranged from 4.42 (BthTX-I) to 8.02 (MjTX-I) kcal/mole, compared with 6.47 and 6.88 kcal/mole for trypsin and RNAse, respectively. The values for deltaGD(H)(2)(O) and D1/2 showed that BthTX-I is the least stable among the four myotoxins assayed, with a D1/2 close to that of RNAse, while MjTX-II is conformationally the most stable. Monitoring of the unfolding of RNAse and PrTX-I by a 0 to 6 M urea gradient PAGE revealed transitions from the native (N) to the unfolded (U) state with deltaG(N-U)of 0.22 and 0.41 kcal/mole, respectively. Sigmoidal curves showed well-defined two-stage transitions for both proteins.
Brito
Ana G
AG
Departamento de Bioquímica e Imunologia, FMRP - USP, 14049-900 Ribeirão Preto, SP, Brazil.
Soares
Andreimar M
AM
Homsi-Brandeburgo
Maria I
MI
Borges
Márcia H
MH
Giglio
José R
JR
Penha-Silva
Nilson
N
D000818:Animals; D017837:Bothrops / Q000378:metabolism*; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry*; D010741:Phospholipases A / Q000737:chemistry* / Q000633:toxicity; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry; D013056:Spectrophotometry, Ultraviolet; D013816:Thermodynamics; D014357:Trypsin / Q000737:chemistry; D014508:Urea / Q000737:chemistry*
D014508:Urea; D010741:Phospholipases A; D012259:Ribonuclease, Pancreatic; D014357:Trypsin; D019791:Guanidine