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EFFECT OF TWO POLAR ORGANIC‐AQUEOUS SOLVENT SYSTEMS ON THE STRUCTURE‐FUNCTION RELATIONSHIP OF PROTEASES I. PEPSIN
Author(s) -
KANG YOUNG,
MARANGONI ALEJANDRO G.,
YADA RICKEY Y.
Publication year - 1993
Publication title -
journal of food biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.507
H-Index - 47
eISSN - 1745-4514
pISSN - 0145-8884
DOI - 10.1111/j.1745-4514.1993.tb00480.x
Subject(s) - chemistry , aqueous solution , sodium formate , solvent , chromatography , pepsin , formate , phosphate buffered saline , ethanol , buffer solution , sodium , acetonitrile , nuclear chemistry , inorganic chemistry , enzyme , biochemistry , organic chemistry , catalysis
The effect of various organic‐aqueous solvent systems on the kinetic parameters, intrinsic spectral properties, thermal stability, and proteolytic patterns of porcine pepsin were studied. Two substrates, Z‐His‐Phe(‐NO 2 )‐Phe‐OMe and sodium (Na‐) caseinate, were chosen. Three different buffer compositions were used in the investigation: (1) the standard buffers (20 mM formate buffers, pH 2.1 and 10 mM phosphate buffer, pH 5.7); (2) 5% (v/v) ethanol (EtOH) in the standard buffers; (3) 5% (v/v) acetonitrile (ACN) in the standard buffer. Relative to pepsin in formate buffer (pH 2.1), the K m for Z‐His‐Phe(‐NO 2 )‐Phe‐OMe in 5% EtOH increased from 0.57 mM to 1.03 mM (p ≤ 0.05), while no significant difference was observed in 5% ACN (p > 0.05). The solvent‐induced decrease in V max was much larger in 5% ACN than in 5% EtOH, from 48.0 nmoles/min.mg to 12.3 nmoles/min.mg (p > 0.05) and 35.0 nmoles/min.mg (p > 0.05), respectively, as compared to standard buffer. Relative to pepsin in 10 mM phosphate buffer (pH 5.7), the K m for Na‐caseinate in both 5% EtOH and 5% ACN increased from 4.1 mg/ml to 7.8 mg/ml and 6.2 mg/ml (p ≤ 0.05), respectively while only 5% ACN caused a significant decrease in V max compared with standard buffer, from 11.8μmoles/min.mgto 7.6 μmoles/min.mg (p ≤ 0.05). Changes in kinetic parameters generally corresponded with solvent‐induced structural changes as evidenced by circular dichroism (CD) spectroscopy, a low K m corresponding to low ellipticity in the near‐UV CD spectra (240–320 nm) possibly indicative of a greater protein flexibility. The above results were attributed to differences in properties other than polarity of the solvent systems since the polarity of both 5% EtOH and 5% ACN solutions, as measured by E T (30) scale, was the same. Differential scanning calorimetric studies of pepsin in the different solvent systems showed destabilization of the enzyme in the organic solutions relative to standard buffer, i.e., lowered temperature of denaturation. Altered specificity of pepsin for Na‐caseinate hydrolysis in the presence of the various organic solvents was demonstrated in SDS‐PAGE peptide maps as differences in the banding patterns.