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Biochemical and conformational characterisation of HSP‐3, a stallion seminal plasma protein of the cysteine‐rich secretory protein (CRISP) family
Author(s) -
Magdaleno Leticia,
Gasset Marı́a,
Varea Julio,
Schambony Alexandra M,
Urbanke Claus,
Raida Manfred,
Töpfer-Petersen Edda,
Calvete Juan J
Publication year - 1997
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(97)01514-7
Subject(s) - circular dichroism , chemistry , monomer , molecular mass , isothermal microcalorimetry , cysteine , protein secondary structure , denaturation (fissile materials) , biochemistry , enthalpy , crystallography , microbiology and biotechnology , biology , nuclear chemistry , organic chemistry , physics , quantum mechanics , enzyme , polymer
HSP‐3 is a member of the cysteine‐rich secretory protein (CRISP) family from stallion seminal plasma. We report a large‐scale purification protocol for native HSP‐3. This protein is a non‐glycosylated polypeptide chain with a p I of 8–9 and an isotope‐averaged molecular mass of 24 987±3 Da. The molecular mass of HSP‐3, determined by equilibrium sedimentation, is 26 kDa, showing that the protein exists in solution as a monomer. The concentration of HSP‐3 in the seminal plasma of different stallions ranged from 0.3 to 1.3 mg/ml. On average, 0.9–9 million HSP‐3 molecules/cell coat the postacrosomal and mid‐piece regions of an ejaculated, washed stallion spermatozoon, suggesting a role in sperm physiology. Conformational characterisation of purified HSP‐3 was assessed by combination of circular dichroism and Fourier‐transform infrared spectroscopies and differential scanning microcalorimetry. Based on secondary structure assignment, HSP‐3 may belong to the α+β class of proteins. Thermal denaturation of HSP‐3 is irreversible and follows a non‐two state transition characterised by a T m of 64°C, an enthalpy change of 75 kcal/mol, and a van 't Hoff enthalpy of 184 kcal/mol. Analysis of the spectroscopic and calorimetric data indicates the occurrence of aggregation of denatured HSP‐3 molecules and suggests the monomer as the cooperative unfolding unit.