title: ÐÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ðµ Ð¾ÑÐ¾Ð±ÐµÐ½Ð½Ð¾ÑÑÐµÐ¹ ÑÐµÐ»Ð°ÐºÑÐ°ÑÐ¸Ð¸ ÑÐ²Ð¾Ð±Ð¾Ð´Ð½Ð¾Ð³Ð¾ Ð¸ ÑÐ²ÑÐ·Ð°Ð½Ð½Ð¾Ð³Ð¾ FAD Ð¼ÐµÑÐ¾Ð´Ð¾Ð¼ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ½Ð¾Ð¹ Ð¿Ð¾Ð»ÑÑÐ¸Ð·Ð°ÑÐ¸Ð¾Ð½Ð½Ð¾Ð¹ ÑÐ¿ÐµÐºÑÑÐ¾ÑÐºÐ¾Ð¿Ð¸Ð¸ Ñ Ð²ÑÑÐ¾ÐºÐ¸Ð¼ Ð²ÑÐµÐ¼ÐµÐ½Ð½ÑÐ¼ ÑÐ°Ð·ÑÐµÑÐµÐ½Ð¸ÐµÐ¼
title: Ð²ÑÐ¿ÑÑÐºÐ½Ð°Ñ ÐºÐ²Ð°Ð»Ð¸ÑÐ¸ÐºÐ°ÑÐ¸Ð¾Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¼Ð°Ð³Ð¸ÑÑÑÐ°
titleType: Subtitle
title: Investigation of the characteristic of free and bound FAD relaxation by fluorescence polarizationspectroscopy with high temporal resolution
Creators: name: Ð¯ÑÐºÐ¾Ð², ÐÐ¼Ð¸ÑÑÐ¸Ð¹
Publisher: name: Ð¡Ð°Ð½ÐºÑ-ÐÐµÑÐµÑÐ±ÑÑÐ³ÑÐºÐ¸Ð¹ Ð¿Ð¾Ð»Ð¸ÑÐµÑÐ½Ð¸ÑÐµÑÐºÐ¸Ð¹ ÑÐ½Ð¸Ð²ÐµÑÑÐ¸ÑÐµÑ ÐÐµÑÑÐ° ÐÐµÐ»Ð¸ÐºÐ¾Ð³Ð¾
Year: 2024
Subjects: Ð¡Ð¿ÐµÐºÑÑÐ¾ÑÐºÐ¾Ð¿Ð¸Ñ
ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ñ
ÑÐ»Ð°Ð²Ð¸Ð½-Ð°Ð´ÐµÐ½Ð¸Ð½-Ð´Ð¸Ð½ÐºÑÐ»ÐµÐ¾ÑÐ¸Ð´
Ð»Ð¸Ð¿Ð¾Ð°Ð¼Ð¸Ð´-Ð´ÐµÐ³Ð¸Ð´ÑÐ¾Ð³ÐµÐ½Ð°Ð·Ð°
Ð´Ð²ÑÑ ÑÐ¾ÑÐ¾Ð½Ð½Ð¾Ðµ Ð²Ð¾Ð·Ð±ÑÐ¶Ð´ÐµÐ½Ð¸Ðµ
Ð²ÑÐµÐ¼Ñ-ÑÐ°Ð·ÑÐµÑÐµÐ½Ð½Ð°Ñ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ñ
Ð¿Ð°ÑÐ°Ð¼ÐµÑÑÑ Ð·Ð°ÑÑÑ Ð°Ð½Ð¸Ñ Ð°Ð½Ð¸Ð·Ð¾ÑÑÐ¾Ð¿Ð½Ð¾Ð¹ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸
fluorescence
flavin adenine dinucleotide
lipoamide dehydrogenase
two-photon excitation
time-resolved fluorescence
anisotropic fluorescence decay parameters

description: ÐÐ°Ð½Ð½Ð°Ñ ÑÐ°Ð±Ð¾ÑÐ° Ð¿Ð¾ÑÐ²ÑÑÐµÐ½Ð° Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸ FAD Ð² ÑÐ²Ð¾Ð±Ð¾Ð´Ð½Ð¾Ð¹ Ð¸ ÑÐ²ÑÐ·Ð°Ð½Ð½Ð¾Ð¹ Ñ ÑÐµÑÐ¼ÐµÐ½ÑÐ¾Ð¼ Ð»Ð¸Ð¿Ð¾Ð°Ð¼Ð¸Ð´ Ð´ÐµÐ³Ð¸Ð´ÑÐ¾Ð³ÐµÐ½Ð°Ð·Ð° (LipDH), Ð¿ÑÐ¸ Ð¾Ð´Ð½Ð¾- Ð¸ Ð´Ð²ÑÑÑÐ¾ÑÐ¾Ð½Ð½Ð¾Ð¼ Ð²Ð¾Ð·Ð±ÑÐ¶Ð´ÐµÐ½Ð¸Ð¸ ÑÐµÐ¼ÑÐ¾ÑÐµÐºÑÐ½Ð´Ð½ÑÐ¼Ð¸ Ð»Ð°Ð·ÐµÑÐ½ÑÐ¼Ð¸ Ð¸Ð¼Ð¿ÑÐ»ÑÑÐ°Ð¼Ð¸ Ð² Ð´Ð¸Ð°Ð¿Ð°Ð·Ð¾Ð½Ðµ Ð´Ð»Ð¸Ð½ Ð²Ð¾Ð»Ð½ 360â450 Ð½Ð¼ Ð¸ 720â890 Ð½Ð¼. Ð¦ÐµÐ»ÑÑ ÑÑÐ¾Ð¹ ÑÐ°Ð±Ð¾ÑÑ ÑÐ²Ð»ÑÐµÑÑÑ ÑÑÑÐ°Ð½Ð¾Ð²Ð»ÐµÐ½Ð¸Ðµ Ð¾ÑÐ¾Ð±ÐµÐ½Ð½Ð¾ÑÑÐµÐ¹ Ð¿ÑÐ¾ÑÐµÑÑÐ¾Ð² ÑÐµÐ»Ð°ÐºÑÐ°ÑÐ¸Ð¸ ÑÐ²Ð¾Ð±Ð¾Ð´Ð½Ð¾Ð³Ð¾ FAD Ð¸ ÑÐ²ÑÐ·Ð°Ð½Ð½Ð¾Ð³Ð¾ Ñ ÑÐµÑÐ¼ÐµÐ½ÑÐ¾Ð¼ LipDH. ÐÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ Ð¿ÑÐ¾Ð²Ð¾Ð´Ð¸Ð»Ð¸ÑÑ Ð¼ÐµÑÐ¾Ð´Ð¾Ð¼ Ð²ÑÐµÐ¼Ñ-ÑÐ°Ð·ÑÐµÑÐµÐ½Ð½Ð¾Ð¹ Ð¿Ð¾Ð»ÑÑÐ¸Ð·Ð°ÑÐ¸Ð¾Ð½Ð½Ð¾Ð¹ Ð»Ð°Ð·ÐµÑÐ½Ð¾Ð¹ ÑÐ¿ÐµÐºÑÑÐ¾ÑÐºÐ¾Ð¿Ð¸Ð¸, Ð² ÑÐ°Ð¼ÐºÐ°Ñ ÐºÐ¾ÑÐ¾ÑÐ¾Ð³Ð¾ ÑÐµÐ³Ð¸ÑÑÑÐ¸ÑÐ¾Ð²Ð°Ð»Ð¸ÑÑ ÑÐ¸Ð³Ð½Ð°Ð»Ñ Ð·Ð°ÑÑÑÐ°Ð½Ð¸Ñ Ð´Ð²ÑÑ Ð¾ÑÑÐ¾Ð³Ð¾Ð½Ð°Ð»ÑÐ½ÑÑ Ð¿Ð¾Ð»ÑÑÐ¸Ð·Ð¾Ð²Ð°Ð½Ð½ÑÑ ÐºÐ¾Ð¼Ð¿Ð¾Ð½ÐµÐ½Ñ Ñ Ð²ÑÐµÐ¼Ñ-ÐºÐ¾ÑÑÐµÐ»ÑÑÐ¸Ð¾Ð½Ð½Ð¾Ð¹ ÑÐ¸ÑÑÐµÐ¼Ñ ÑÑÐµÑÐ° ÑÐ¾ÑÐ¾Ð½Ð¾Ð². ÐÑÐ»Ð¾ Ð¾Ð±Ð½Ð°ÑÑÐ¶ÐµÐ½Ð¾, ÑÑÐ¾ FAD, ÑÐ²ÑÐ·Ð°Ð½Ð½ÑÐ¹ Ñ ÑÐµÑÐ¼ÐµÐ½ÑÐ¾Ð¼ LipDH ÑÐ°ÑÐ°ÐºÑÐµÑÐ¸Ð·ÑÐµÑÑÑ Ð´Ð²ÑÐ¼Ñ Ð²ÑÐµÐ¼ÐµÐ½Ð°Ð¼Ð¸ Ð·Ð°ÑÑÑÐ°Ð½Ð¸Ñ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸: Ï1 = 0.27 Ð½Ñ (a1 = 0.17) Ð¸ Ï2 = 4.34 Ð½Ñ (a2 = 0.83), Ð³Ð´Ðµ Ð² ÑÐºÐ¾Ð±ÐºÐ°Ñ ÑÐºÐ°Ð·Ð°Ð½Ñ Ð²ÐµÑÐ¾Ð²ÑÐµ ÐºÐ¾ÑÑÑÐ¸ÑÐ¸ÐµÐ½ÑÑ. ÐÑÐ¸ ÑÑÐ¾Ð¼ ÑÐ²Ð¾Ð±Ð¾Ð´Ð½ÑÐ¹ FAD ÑÐ°ÑÐ°ÐºÑÐµÑÐ¸Ð·ÑÐµÑÑÑ ÑÐµÑÑÑÑÐ¼Ñ Ð²ÑÐµÐ¼ÐµÐ½Ð°Ð¼Ð¸ Ð·Ð°ÑÑÑÐ°Ð½Ð¸Ñ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸: Ï1 = 20 Ð¿Ñ (a1 = 0.38), Ï2 â¼ 220 Ð¿Ñ (a2 = 0.13), Ï3 = 2.3â2.6 Ð½Ñ (a3= 0.34) Ð¸ Ï4 = 3.7â4.8 Ð½Ñ (a4 = 0.15), Ð³Ð´Ðµ Ð² ÑÐºÐ¾Ð±ÐºÐ°Ñ ÑÐºÐ°Ð·Ð°Ð½Ñ Ð²ÐµÑÐ¾Ð²ÑÐµ ÐºÐ¾ÑÑÑÐ¸ÑÐ¸ÐµÐ½ÑÑ. ÐÑÐ»Ð¾ ÑÑÑÐ°Ð½Ð¾Ð²Ð»ÐµÐ½Ð¾, ÑÑÐ¾ Ð²ÑÐµÐ¼ÐµÐ½Ð° Ï1 Ð¸ Ï2 Ð¾Ð±ÑÑÐ»Ð¾Ð²Ð»ÐµÐ½Ñ Ð¿ÑÐ¾ÑÐµÑÑÐ°Ð¼Ð¸ ÑÐµÐ»Ð°ÐºÑÐ°ÑÐ¸Ð¸ Ð´Ð²ÑÑ ÑÐ°Ð·Ð»Ð¸ÑÐ½ÑÑ ÑÐ»Ð¾Ð¶ÐµÐ½Ð½ÑÑ ÐºÐ¾Ð½ÑÐ¾ÑÐ¼Ð°ÑÐ¸Ð¹ FAD, Ð° Ð²ÑÐµÐ¼Ñ Ï4 Ð¾Ð±ÑÑÐ»Ð¾Ð²Ð»ÐµÐ½Ð¾ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸ÐµÐ¹ ÑÐ°Ð·Ð²ÐµÑÐ½ÑÑÐ¾Ð¹ ÐºÐ¾Ð½ÑÐ¾ÑÐ¼Ð°ÑÐ¸Ð¸ FAD. ÐÐ²Ðµ ÑÐ»Ð¾Ð¶ÐµÐ½Ð½ÑÐµ ÐºÐ¾Ð½ÑÐ¾ÑÐ¼Ð°ÑÐ¸Ð¸ ÑÐ°Ð·Ð»Ð¸ÑÐ°ÑÑÑÑ Ð¿Ð¾Ð»Ð¾Ð¶ÐµÐ½Ð¸ÐµÐ¼ Ð°Ð´ÐµÐ½Ð¸Ð½Ð° Ð½Ð°Ð´ Ð¸Ð·Ð¾Ð°Ð»Ð»Ð¾ÐºÑÐ°Ð·Ð¸Ð½Ð¾Ð²ÑÐ¼ ÐºÐ¾Ð»ÑÑÐ¾Ð¼. Ð­ÐºÑÐ¿ÐµÑÐ¸Ð¼ÐµÐ½ÑÐ°Ð»ÑÐ½Ð¾ Ð¿Ð¾Ð»ÑÑÐµÐ½Ð½Ð¾Ðµ Ð¸Ð·Ð¼ÐµÐ½ÐµÐ½Ð¸Ðµ Ð²ÐµÑÐ¾Ð²ÑÑ ÐºÐ¾ÑÑÑÐ¸ÑÐ¸ÐµÐ½ÑÐ¾Ð² FAD Ð² Ð²Ð¾Ð´Ð½Ð¾-ÑÐ¿Ð¸ÑÑÐ¾Ð²ÑÑ ÑÐ°ÑÑÐ²Ð¾ÑÐ°Ñ Ð¿Ð¾Ð´ÑÐ²ÐµÑÐ¶Ð´Ð°ÐµÑ Ð¼Ð¾Ð´ÐµÐ»Ñ ÑÐ°ÑÐ¿ÑÐµÐ´ÐµÐ»ÐµÐ½Ð¸Ñ ÐºÐ¾Ð½ÑÐ¾ÑÐ¼Ð°ÑÐ¸Ð¹, Ð¿Ð¾ÑÑÐ¸ÑÐ°Ð½Ð½ÑÑ Ð¼ÐµÑÐ¾Ð´Ð¾Ð¼ Ð¼Ð¾Ð»ÐµÐºÑÐ»ÑÑÐ½Ð¾Ð¹ Ð´Ð¸Ð½Ð°Ð¼Ð¸ÐºÐ¸. Ð¢Ð°ÐºÐ¶Ðµ Ð±ÑÐ»Ð¸ Ð¿ÑÐ¾Ð²ÐµÐ´ÐµÐ½Ñ Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ñ Ð°Ð½Ð¸Ð·Ð¾ÑÑÐ¾Ð¿Ð¸Ð¸ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸ ÑÐ²Ð¾Ð±Ð¾Ð´Ð½Ð¾Ð³Ð¾ FAD Ð¾Ñ Ð´Ð»Ð¸Ð½Ñ Ð²Ð¾Ð»Ð½Ñ Ð²Ð¾Ð·Ð±ÑÐ¶Ð´ÐµÐ½Ð¸Ñ Ð¿ÑÐ¸ Ð¾Ð´Ð½Ð¾- Ð¸ Ð´Ð²ÑÑÑÐ¾ÑÐ¾Ð½Ð½Ð¾Ð¼ Ð²Ð¾Ð·Ð±ÑÐ¶Ð´ÐµÐ½Ð¸Ð¸. ÐÐ»Ñ FAD, ÑÐ²ÑÐ·Ð°Ð½Ð½Ð¾Ð³Ð¾ Ñ LipDH, Ð·Ð°ÑÐµÐ³Ð¸ÑÑÑÐ¸ÑÐ¾Ð²Ð°Ð½Ð¾ ÑÑÐ±Ð½Ð°Ð½Ð¾ÑÐµÐºÑÐ½Ð´Ð½Ð¾Ðµ Ð²ÑÐµÐ¼Ñ Ð·Ð°ÑÑÑÐ°Ð½Ð¸Ñ Ð°Ð½Ð¸Ð·Ð¾ÑÑÐ¾Ð¿Ð¸Ð¸ ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ¸Ð¸ Ïrb = 130 Ð¿Ñ. ÐÐ½Ð¾ Ð¾Ð±ÑÑÐ»Ð¾Ð²Ð»ÐµÐ½Ð¾ Ð°Ð½Ð¸Ð·Ð¾ÑÑÐ¾Ð¿Ð½Ð¾Ð¹ ÐºÐ¾Ð»ÐµÐ±Ð°ÑÐµÐ»ÑÐ½Ð¾Ð¹ ÑÐµÐ»Ð°ÐºÑÐ°ÑÐ¸ÐµÐ¹ Ð² Ð²Ð¾Ð·Ð±ÑÐ¶Ð´ÐµÐ½Ð½Ð¾Ð¼ ÑÐ¾ÑÑÐ¾ÑÐ½Ð¸Ð¸ ÐºÐ¾ÑÐµÑÐ¼ÐµÐ½ÑÐ¾Ð², ÐºÐ¾ÑÐ¾ÑÐ°Ñ ÑÐ¾Ð¿ÑÐ¾Ð²Ð¾Ð¶Ð´Ð°Ð»Ð°ÑÑ Ð¾Ð²Ð¾ÑÐ¾ÑÐ¾Ð¼ Ð´Ð¸Ð¿Ð¾Ð»ÑÐ½Ð¾Ð³Ð¾ Ð¼Ð¾Ð¼ÐµÐ½ÑÐ° Ð¿ÐµÑÐµÑÐ¾Ð´Ð° Ð·Ð° ÑÑÐµÑ Ð¿ÐµÑÐµÑÑÑÐ¾Ð¹ÐºÐ¸ ÐºÐ¾Ð½ÑÐ¸Ð³ÑÑÐ°ÑÐ¸Ð¸ ÑÐ´ÐµÑ Ð² Ð¼Ð¾Ð»ÐµÐºÑÐ»Ðµ FAD. ÐÐ¾Ð»ÑÑÐµÐ½Ð½ÑÐµ ÑÐµÐ·ÑÐ»ÑÑÐ°ÑÑ Ð¸Ð¼ÐµÑÑ Ð±Ð¾Ð»ÑÑÑÑ Ð½Ð°ÑÑÐ½ÑÑ Ð·Ð½Ð°ÑÐ¸Ð¼Ð¾ÑÑÑ, ÑÐ°Ðº ÐºÐ°Ðº Ð¾Ð½Ð¸ Ð¼Ð¾Ð³ÑÑ Ð±ÑÑÑ Ð¸ÑÐ¿Ð¾Ð»ÑÐ·Ð¾Ð²Ð°Ð½Ñ Ð´Ð»Ñ Ð¾Ð¿Ð¸ÑÐ°Ð½Ð¸Ñ ÑÐµÐ·ÑÐ»ÑÑÐ°ÑÐ¾Ð² Ð¸ÑÑÐ»ÐµÐ´Ð¾Ð²Ð°Ð½Ð¸Ð¹ Ð¾ÐºÐ¸ÑÐ»Ð¸ÑÐµÐ»ÑÐ½Ð¾-Ð²Ð¾ÑÑÑÐ°Ð½Ð¾Ð²Ð¸ÑÐµÐ»ÑÐ½ÑÑ ÑÐµÐ°ÐºÑÐ¸Ð¸ Ð² ÐºÐ»ÐµÑÐºÐ°Ñ Ð¶Ð¸Ð²ÑÑ Ð¾ÑÐ³Ð°Ð½Ð¸Ð·Ð¼Ð¾Ð² ÑÐ»ÑÐ¾ÑÐµÑÑÐµÐ½ÑÐ½ÑÐ¼Ð¸ Ð¼ÐµÑÐ¾Ð´Ð°Ð¼Ð¸, Ð° ÑÐ°ÐºÐ¶Ðµ Ð´Ð»Ñ ÑÐ°Ð·ÑÐ°Ð±Ð¾ÑÐºÐ¸ Ð½ÐµÐ¸Ð½Ð²Ð°Ð·Ð¸Ð²Ð½ÑÑ Ð¼ÐµÑÐ¾Ð´Ð¾Ð² Ð´Ð¸Ð°Ð³Ð½Ð¾ÑÑÐ¸ÐºÐ¸ ÑÐ¾ÑÐ¸Ð°Ð»ÑÐ½Ð¾-Ð·Ð½Ð°ÑÐ¸Ð¼ÑÑ Ð·Ð°Ð±Ð¾Ð»ÐµÐ²Ð°Ð½Ð¸Ð¹ Ð½Ð° ÑÐ°Ð½Ð½Ð¸Ñ ÑÑÐ°Ð´Ð¸ÑÑ.
description: This work focuses on the study of flavin adenine dinucleotide (FAD) fluorescence in free and protein-bound lipoamide dehydrogenase (LipDH), under single- and two-photon excitation using femtosecond laser pulses in the wavelength range 360â450 nm and 720â890 nm, respectively. The aim of the research is to investigate the characteristics of relaxation processes associated with LipDH and free FAD, using the method of high-resolution polarization laser spectroscopy. Two orthogonal fluorescence components are recorded using a photon-counting system with time-correlated measurements. It was found that LipDH-bound FAD exhibits two characteristic fluorescence decay times, Ï1 = 0.27 ns (a1 = 0.17) and Ï2 = 4.34 ns (a2 = 0.83). Free FAD, on the other hand, exhibits four decay times: Ï1 = 20 ps (a1 =0.38), Ï2 â¼ 220 ps (a2 = 0.13), Ï3 = 2.3â2.6 ns (a3 = 0.34) and Ï4 = 3.7â 4.8 ns (a4 = 0.15). It has been determined that the times Ï1 and Ï2 are associated with the relaxation processes of two distinct folded FAD conformations, while the time Ï4 is related to the fluorescence from the unfolded FAD structure. The two folded conformations differ in terms of the position of the adenine relative to the isoalloxazine ring. The experimentally determined change in the FAD weighting coefficients in aqueous alcoholic solutions confirms the conformational distribution model predicted by the molecular dynamics simulation. Additionally, the anisotropy of fluorescence from free FAD excited at different wavelengths has been studied using single- and two-photon excitation. For FAD bound to LipDH, a fluorescence anisotropy decay time of Ïrb = 130 ps. These results are of significant scientific importance, as they can be utilized to describe the outcomes of redox reaction studies in living cells using fluorescent techniques, as well as for the development of non-invasive techniques for early diagnosis of socially significant diseases.