Влияние рH на извлечение белков из поджелудочной железы Sus scrofa

Поджелудочная железа (ПЖ) свиней содержит множество ферментов, структурных, регуляторных, секреторных, рецепторных и других биологически активных веществ, обеспечивающих как функционирование органа, так и его биологическую роль в организме. Целью настоящей работы было изучение влияния изменения pH в 0,9%-м растворе хлорида натрия, использующегося в качестве экстрагента, на эффективность извлечения биологически активных веществ белковой природы из поджелудочной железы свиней. Экстракцию проводили 0,9% NaCl, 0,9% NaCl pH≤4 и 0,9% NaCl, pH=8,5 со скоростью перемешивания 400 об/мин в течение 150 мин при 4 ºС, соотношение ПЖ: экстрагент 1:5, супернатант отделяли центрифугированием. Концентрация белка была измерена биуретовой реакцией на полуавтоматическом биохимическом анализаторе Biochem SA. Протеомный состав экстрактов и нативной поджелудочной железы оценивали методом одномерного электрофореза по Лэммли в 10% полиакриламидном геле в камере VE-10. Полные цифровые изображения электрофореграмм получали с помощью сканера Bio-5000 Plus, затем их редактировали в графическом редакторе и анализировали с помощью программного обеспечения ImageJ. При определении интенсивности белковых фракций отмечалось, что применение 0,9% NaCl способствовало большему выходу в экстрагент белков с молекулярными массами (Мм) 200 кДа, 150 кДа, 69 кДа, 52 кДа и 33 кДа, смещение рН в кислую область стимулировало выход фракций с молекулярными массами 130 кДа, 50 кДа, 49 кДа, 45 кДа, 40 кДа, 30 кДа и 27 кДа, а в щелочную — только 47 кДа и 42 кДа. Большинство протеолитических ферментов ПЖ имеет молекулярную массу в диапазоне 34–23 кДа, за исключением незрелой формы карбоксипептидаз с Мм 45–47 кДа, на полученных электрофореграммах наибольшая интенсивность белковых полос выражена в области с Мм менее 33 кДа. Также отмечалось присутствие интенсивных белковых фракций в области молекулярных масс менее 50–52 кДа и 40 кДа, что может соответствовать таким энзимам, как панкреатическая липаза и фосфолипаза А2, и наличие белковых фракций с Мм выше 130 кДа, соответствующих различным типам и изоформам коллагена и ламинина. Кроме того, такие процессы, как белковая агрегация и протеолиз также могут оказывать влияние на молекулярно-массовое распределение белковых фракций.

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