Ice recrystallization inhibition activity varies with ice-binding protein type and does not correlate with thermal hysteresis

Cryobiology. 2021 Apr:99:28-39. doi: 10.1016/j.cryobiol.2021.01.017. Epub 2021 Jan 30.


Ice-binding proteins (IBPs) inhibit the growth of ice through surface adsorption. In some freeze-resistant fishes and insects, circulating IBPs serve as antifreeze proteins to stop ice growth by lowering the freezing point. Plants are less able to avoid freezing and some use IBPs to minimize the damage caused in the frozen state by ice recrystallization, which is the growth of large ice grains at the expense of small ones. Here we have accurately and reproducibly measured the ice recrystallization inhibition (IRI) activity of over a dozen naturally occurring IBPs from fishes, insects, plants, and microorganisms using a modified 'splat' method on serial dilutions of IBPs whose concentrations were determined by amino acid analysis. The endpoint of IRI, which was scored as the lowest protein concentration at which no recrystallization was observed, varied for the different IBPs over two orders of magnitude from 1000 nM to 5 nM. Moreover, there was no apparent correlation between their IRI levels and reported antifreeze activities. IBPs from insects and fishes had similar IRI activity, even though the insect IBPs are typically 10x more active in freezing point depression. Plant IBPs had weak antifreeze activity but were more effective at IRI. Bacterial IBPs involved in ice adhesion showed both strong freezing point depression and IRI. Two trends did emerge, including that basal plane binding IBPs correlated with stronger IRI activity and larger IBPs had higher IRI activity.

Keywords: Antifreeze glycoprotein; Antifreeze protein; Cryopreservation; Ice crystal; Ice recrystallization inhibition.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antifreeze Proteins / metabolism
  • Carrier Proteins*
  • Cryopreservation / methods
  • Crystallization
  • Fishes
  • Freezing
  • Ice*
  • Insecta


  • Antifreeze Proteins
  • Carrier Proteins
  • Ice

Grants and funding