Multiple forms of acidic endopeptidase from germinated barley.

Area of Science:

• Biochemistry
• Enzymology
• Plant Science

Background:

• Endopeptidases play vital roles in protein hydrolysis.
• Germinated barley is a source of various proteolytic enzymes.
• Understanding barley endopeptidase characteristics is important for agricultural and food science applications.

Purpose of the Study:

• To purify and characterize endopeptidases from germinated barley.
• To investigate the enzymatic properties, including cofactor dependence and substrate specificity.
• To determine the molecular weight and isoelectric points of the purified enzymes.

Main Methods:

• Purification using affinity chromatography and density-gradient electrofocusing.
• Molecular weight determination via gel filtration (Sephadex G-75).
• Enzyme activity assays with N,N-dimethylhemoglobin as substrate at pH 3.8.

Main Results:

• The endopeptidase preparation consisted of three to four components with isoelectric points (pI) of 4.15, 4.28, and 4.37.
• The major component had a molecular weight of 14,100.
• Enzyme activity was sulfhydryl-dependent, requiring cysteine, and was sensitive to oxygen.
• Narrow concentration tolerances were observed for cysteine and substrate.
• Kinetic parameters (Km and Vmax) were determined for N,N-dimethylhemoglobin.

Conclusions:

• The purified barley endopeptidases are sulfhydryl-dependent enzymes with specific molecular and biochemical properties.
• The enzymes are sensitive to oxygen and exhibit narrow substrate and cofactor concentration tolerances.
• The characterization provides insights into the function and regulation of barley endopeptidases, ruling out autolytic artifacts.