C: Food Chemistry Thermal Denaturation of Tilapia Myosin and Its Subunits as Affected by Constantly Increasing Temperature Zachary Harold Reed, William Guilford, and Jae Won Park Abstract: Puriﬁed tilapia myosin was digested with α-chymotrypsin and puriﬁed to obtain heavy meromyosin (HMM) and light meromyosin (LMM). Biochemical properties of tilapia myosin, HMM, and LMM were characterized. Surface hydrophobicity (S o ) showed an increase for myosin and HMM between 30 and 40 ◦ C and reached a plateau at 70 ◦ C. LMM, in a small magnitude, also showed a continuous increase to 70 ◦ C. Total sulfhydryl content (TSH) demonstrated that the SH residue content of HMM was nearly double that of LMM. Surface reactive sulfhydryl groups (SRSH) for myosin and HMM were relatively unchanged from 10 to 30 ◦ C but increased from 30 to 50 ◦ C. The exposure of buried hydrophobic and sulfhydryl groups of myosin and HMM increased as the myosin and HMM were constantly heated. However, the TSH and SRSH results indicated that the stability of LMM was likely due to its α-helix conformation. Reducing and nonreducing sodium dodecylsulfate-polyacryamide gel electrophoresis helped to understand the role of disulﬁde bonds in thermal aggregation of tilapia myosin, HMM, and LMM Keywords: constant heating, HMM, LMM, myosin, tilapia Introduction Myosin is the major muscle protein that is found in ﬁsh and compromises approximately 55% to 60% of the myoﬁbrillar pro- teins found in ﬁsh (Lanier and others 2005). The molecular weight of myosin heavy chain when dissociated in strong denaturing solu- tions is approximately 220 kDa. When subjected to digestion using α-chymotrypsin, myosin can be fragmented into 2 main portions, namely heavy meromyosin (HMM) and light meromyosin (LMM) (Szent-G ¨ orgyi 1953; Lowey and Cohen 1962; Lowey and others 1969). HMM contains the globular head unit along with a short portion of the tail, and the LMM is made up of the α-helical coiled-coil tail with molecular weights of 350 and 125 kDa, re- spectively, for the dimers (Margossian and Lowey 1982). In order to better understand the important roles that myosin, HMM, and LMM play in the gelation of myoﬁbrillar proteins, extensive studies have been performed on puriﬁed myosin and its fragments from a variety of ﬁsh species such as Alaska pollock (Togashi and others 2002), sardine (Ogawa and others 1999a), Japanese stingﬁsh (Nagai and others 1999), carp (Tsuchiya and Matsumoto 1975), cod, and herring (Chan and others 1993). By studying myosin and its subfragments it is possible to elucidate the role that each fragment plays in myosin aggregation during heating. Chan and others (1993) studied the aggregation of HMM and LMM subfragments from cod and herring, and found that at 30 to 40 ◦ C an initial aggregate formed by the interaction of the HMM portion of myosin. This was followed by compound aggregates being formed at 40 to 55 ◦ C by the interaction of LMM. Reed MS 20110317 Submitted 3/11/2011, Accepted 6/29/2011. Authors Reed and Park are with Seafood Research and Education Center, Oregon State Univ., 2001 Marine Drive Rm 253, Astoria, OR 97103, U.S.A. Author Guilford is with Department of Biomedical Engineering, Univ. of Virginia, Charlottesville, VA 22908, U.S.A. Direct inquiries to author Park (E-mail: jae.park@oregonstate.edu). and Park (2011) found that α-chymotryptic digestion of tilapia myosin, to form HMM and LMM, allowed for important insights into the thermal stability and gelation properties of tilapia. In this study we attempted to elucidate the thermal denatura- tion of tilapia myosin, heavy meromyosin, and light meromyosin as affected by constant heating. Puriﬁed myosin, HMM, and LMM were constantly heated in order to understand the biochemical changes that took place. Surface hydrophobicity, surface reactive sulfhydryl, and total sulfhydryl content were investigated to deter- mine the role that heat treatment plays on the thermal denatura- tion/aggregation of tilapia myosin and its subfragments. Materials and Methods Fish Live ﬁsh were killed and gutted at a supermarket in Portland, Oreg., and was transported on ice to the Oregon State Univ. Seafood Research and Education Center (Astoria, Oreg., U.S.A.). Tilapia was ﬁlleted, skinned, and cut into strips. A Kitchenaid stand mixer (St. Joseph, Mich., U.S.A.) with a food grinder and grind plate (prechilled to 4 ◦ C) was used to grind the tilapia strips. Estimated postmortem time before extraction was 4 h. It is gen- erally known that pre-rigor muscle is more effective for obtaining puriﬁed muscle myosin (Connell 1962; Trucco and others 1982; Park 1988) Myosin preparation Myosin was puriﬁed according to the method of Martone and others (1986) with slight modiﬁcations. All steps in the prepara- tion of myosin were carried out in a 4 ◦ C cold room and samples were stored in an ice bath during all stirring steps. Tilapia was mixed with 10 volumes (1:10) of solution A (0.1 M KCl, 1 mM phenylmethylsulfonyl ﬂuoride [PMSF], 0.02% NaN 3 , and 20 mM Tris–HCl, pH 7.5) and homogenized at speed 1 (Powergen 700, Fisher Scientiﬁc, Pittsburgh, Pa., U.S.A.) for 1 min, stirred for C  2011 Institute of Food Technologists R  C1018 Journal of Food Science  Vol. 76, Nr. 7, 2011 doi: 10.1111/j.1750-3841.2011.02326.x Further reproduction without permission is prohibited