June 2017
Volume 58, Issue 8
Open Access
ARVO Annual Meeting Abstract  |   June 2017
αA-crystallin derived mini chaperone stabilizes bovine γ-crystallin aggregation by affecting its zeta (ζ) potential.
Author Affiliations & Notes
  • Ashutosh Phadte
    Biochemistry, University of Missouri, Columbia, Missouri, United States
    Ophthalmology, University of Missouri, Columbia, Missouri, United States
  • Puttur Santhoshkumar
    Ophthalmology, University of Missouri, Columbia, Missouri, United States
  • K Krishna Sharma
    Ophthalmology, University of Missouri, Columbia, Missouri, United States
    Biochemistry, University of Missouri, Columbia, Missouri, United States
  • Footnotes
    Commercial Relationships   Ashutosh Phadte, None; Puttur Santhoshkumar, None; K Krishna Sharma, None
  • Footnotes
    Support  NH EY023219
Investigative Ophthalmology & Visual Science June 2017, Vol.58, 5299. doi:
  • Views
  • Share
  • Tools
    • Alerts
      ×
      This feature is available to Subscribers Only
      Sign In or Create an Account ×
    • Get Citation

      Ashutosh Phadte, Puttur Santhoshkumar, K Krishna Sharma; αA-crystallin derived mini chaperone stabilizes bovine γ-crystallin aggregation by affecting its zeta (ζ) potential.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5299.

      Download citation file:


      © ARVO (1962-2015); The Authors (2016-present)

      ×
  • Supplements
Abstract

Purpose : Post-translational modifications such as oxidation on γ-crystallins have been implicated in the development of age related cataracts. Our past studies have showed that αA-crystallin–derived mini-chaperone (mini-αA) inhibits aggregation of bovine γ-crystallin. To understand the mechanisms for this effect, we investigated changes in the surface charge (zeta [ζ] potential) responsible for maintaining the solubility of γ-crystallin under oxidative conditions, and determined the effect of mini-αA on the ζ potential of aggregating γ-crystallin.

Methods : H2O2 induced aggregation of γ-crystallin (250μg) was monitored at 360nm in a Spectramax i3 Multimode Microplate detection platform (Molecular Devices, Sunnyvale, CA). For ζ potential measurements, 1mg bovine γ-crystallin aliquots were incubated in the presence of 100μM H2O2 for 3 and 6 hours at 37°C, either alone or with 200μg of mini-αA chaperone peptide. Zeta measurements were taken at different time points corresponding to the aggregation pattern of γ-crystallin in a Nanobrook Omni Zeta potential analyzer (Brookhaven Instruments, Holtsville, NY).

Results : Native bovine γ-crystallin showed an average ζ value of -15.96±1.1 mV. The ζ potential dropped to -13.90±2.40 mV and to -9.60±1.51mV respectively, after 3 and 6 hours of incubation with 100Μm H2O2 at 37°C. Interestingly, increased light scattering at 360nm was observed at each of these time points. Such a change in the ζ potential to a value approaching zero indicates sample instability and subsequent aggregation. With the addition of mini-αA (ζ=-19.64±2.15Mv), the ζ potential value of γ-crystallin was -18.63±0.23 mV at 3 hours and -22.30±2.70 mV at 6 hrs. These time points coincided with minimal light scattering of H2O2 treated γ-crystallin at 360nm, suggesting that the peptide prevents γ-crystallin aggregation by compensating its loss of surface charge.

Conclusions : Changes in γ-crystallin stability are accompanied by a changes in its ζ potential. Protein aggregation is known to be associated with a ζ value approaching zero. Stabilization of γ-crystallin by mini-αA is through compensation of the lost surface charges, and the resultant γ-crystallin - mini-αA complex exhibits a higher ζ potential value. The data show that ζ potential measurements can be used to effectively monitor changes in protein stability.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

×
×

This PDF is available to Subscribers Only

Sign in or purchase a subscription to access this content. ×

You must be signed into an individual account to use this feature.

×