Heat stability of plasmin (milk proteinase) and plasminogen

Abstract

The effect of heating on plasmin activity in various media, including phosphate buffer pH 7-0, skim milk, blood plasma, solutions of casein and solutions of whey proteins were investigated. Plots of log residual activity v. heating time were linear at all temperatures from 63 to 143 °C. In buffer solutions the presence of casein led to substantial substrate protection, the Arrhenius plots being linear both in the presence and absence of casein. The activation energy, E a , for the inactivation reaction, was 62-4 kj/mol in buffer alone and 58-4 kJ/mol with casein present at 25 mg/ml. In skim milk, despite the presence of casein at a similar concentration, plasmin was no more stable to heat than in buffer alone, and a curved Arrhenius plot was obtained indicating a more complex inactivation mechanism. Heating in the presence of proteins having free -SH groups accelerated the inactivation of plasmin. The role of -SH groups was confirmed by experiments with added a-lactalbumin, in which no free -SH groups occur, and reduced carboxymethylated /?-lactoglobulin, both of which were without effect. In blood plasma, plasmin was less stable to heat than in buffer (pH 7 -0) or in skim milk. Plasminogen behaved very similarly to plasmin either when activated to plasmin with urokinase before heating or when activated afterwards. A hypothesis is presented to describe the heat inactivation and denaturation of plasmin. Technologically important findings are that in skim milk plasmin was largely unaffected by pasteurization conditions and 30-40% of its activity remained even after ultra high temperature processing conditions.

Effects of cysteine addition and heat treatment during non-fat dry milk processing on the plasmin enzyme system

“…This observation is likely due to the denaturation of whey proteins at high temperatures, specifically β-lactoglobulin [21]. The unfolding of β-lactoglobulin exposes a free sulfhydryl group that binds to PL and PG via sulfhydryl/disulfide interchanges, thus rendering it inactive, which has been demonstrated by several authors [1,18,26]. The addition of cysteine decreased PL and PGderived activities without higher heat treatments, as in the LH fluid milk and NFDM, because cysteine initiates thiol-disulfide interchange reactions, resulting in polymerization of PG or binding of PL and PG with other free sulfhydryls in milk proteins.…”

Barostability of milk plasmin activity

“…The results concerning the effects of milk proteins on plasmin stability are similar to previous observations on the heat inactivation mechanism of plasmin. Specifically, several studies have reported significant protection of plasmin in the presence of sodium caseinate, and enhanced inactivation in the presence of β-lactoglobulin [1,22,29]. Heat inactivation of plasmin in the presence of β-lactoglobulin is thought to be linked to the formation of thiol-disulphide bonds with unfolded β-lactoglobulin [13,16].…”

“…Casein in milk increases the thermal stability of plasmin [1]. The mechanisms and kinetics of thermal inactivation of plasmin have been extensively studied [1,16,22].…”

Mastitis impact on technological properties of milk and quality of milk products—a review

“…Minus and plus symbols indicate an inhibition and an activation, respectively. Question marks indicate that the relationship is suspected but not clearly demonstrated 30% to 40% of plasmin activity can be detected in the milk (Alichanidis et al 1986). Proteolysis of β-casein by plasmin has been shown to play a role in cheese flavor enhancement (Farkye and Landkammer 1992;Farkye and Fox 1992).…”