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Role of Sulfhydryl Groups in the Activity of Milk Lipase¹

Department of Dairy Science, University of Nebraska, Lincoln

ABSTRACT

Studies were conducted to investigate whether purified milk lipase contained -SH groups and whether such groups were associated with the active centers of the purifled milk lipase. Rates of interaction of para-chloromercuribenzoate with the enzyme, determined spectrophotometrically and enzymically, were of the same order, indicating the presence of essential -SH groups in the enzyme.

Para-chloromercuribenzoate, iodoacetic acid, and formamidine disulflde inhibited the enzyme completely, whereas sodium arsenite and hydrogen peroxide inhibited it only partially. N-ethyl maleimide, a specific reagent for free -SH groups only, was only partially inhibitory at all the concentrations investigated. The data indicated the presence of one free and one masked -SH group in the lipase molecule. Dialysis of the lipase inhibited by the various -SH blocking reagents or the addition of glutathione or mercaptoethanol to the dialyzed enzyme did not regenerate the enzyme.

Glutathione stimulated the lipase activity, whereas mercaptoethanol and cysteine hydrochloride inhibited its activity. However, both glutathione and mercaptoethanol partially stabilized the enzyme during storage at 30 and 37 C. Propyl gallate stabilized the enzyme at 30 C hut not at 37 C, whereas alphatocopherol destabilized the enzyme at both temperatures.

¹ Published with the approval of the Director as paper no. 1775, Journal Series, Nebraska Agricultural Experiment Station, Lincoln.