Mercury-binding proteins in tuna and salmon

Dietary consumption of fish is considered as one of the major pathways for human intake of mercury (Hg). The bioavailability and toxicity of Hg depends on the its speciation. In general, around 90 % of the Hg present in marine fish is in the form of methylmercury (MeHg). Methylmercury is known to form adducts with some proteins, however, the information about the mercury-binding proteins in fish samples is limited.

The new study:
The Chinese group of researchers used size-exclusion chromatography (SEC) hyphenated with inductively coupled plasma-mass spectrometry (ICP-MS) to identify mercury-binding proteins in Hg-contaminated tuna and salmon fish.  Tuna fish samples were obtained from Japan, Vietnam, Indonesia, and the Marshall Islands. Salmon fish samples were obtained from Denmark, Norway, and Chile. The total Hg concentration in tuna muscle samples from Indonesia was the highest, with a mean 317.4 ng/g while tuna from Vietnam was about 6 times lower. Total mercury in salmon was much lower with levels up to 28.4 ng/g in fish from Denmark while salmon from Chile contained only 4.4 ng/g Hg.

Fish muscle samples were mixed with the protein extraction lysis RIPA buffer and lysed at 100 rpm for 1 min with three cycles. Fish muscle samples were extracted on ice for 60 min, followed by centrifugation at 4 °C and 12,000 g for 30 min. The top floating layer in centrifuge tubes constituted fat, and the precipitate was cell debris. The solution in the middle layer contained the soluble proteins. The extracted proteins were separated by SEC with metal detection by ICP-MS. The chromatographic fractions containing Hg-binding proteins were collected using a split-flow valve for further analysis by ESI-MS.

The fractions containing Hg- and other metal-binding proteins were mixed with the gel loading buffer and electrophoresed. Gel bands were incubated with sequencing-grade modified trypsin and digested overnight at 37 °C. The trypsin digested peptides were analysed by ESI-MS. The Hg-binding protein in the electrophoresis band with the highest mercury concentration was identified as beta-actin. The concentration of Hg bound to beta-actin accounted for 33 - 70 % of the total Hg in the soluble protein fraction and to 30.2-37.6 % of the total Hg in the fish muscle.  Interestingly, this protein was also binding other metals including Ag, Bi, and Cu. Beta-actin contains five cysteine residues in its amino acid sequence, and the authors speculated that these residues were probably the main sites for interaction between beta-actin and Hg.

The authors concluded that the protein-bound Hg could transform into MeHgCys via gastrointestinal digestion after dietary consumption of fish muscles, which poses health risks to humans.

Used techniques and instrumentation:

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last time modified: September 19, 2024