The gallery is campaigning to keep Raphael's Madonna of the Pinks in the UK after the Duke of Northumberland accepted a £32m offer from the Getty Museum in Los Angeles.
But the duke said there have been attempts to "shame" him into selling the painting to a UK buyer, which has made him reluctant to lend art to the gallery in the future.
"We are very upset about some of the National Gallery claims," a spokesman for the Duke told BBC News Online.
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Owners of great works of art who need to sell should not be forced or shamed into settling for anything less than full value
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Duke of Northumberland
The Madonna of the Pinks has been on loan to the National Gallery for 10 years, and the gallery said it had a deal with the previous duke that would have given it first option on the painting.
But the duke said neither side has evidence of such an agreement.
He was also angered by stories saying proceeds of the sale would go towards a garden renovation, and that the painting had been hanging forgotten in a dusty corridor until a National Gallery expert spotted it.
Writing in the Telegraph, the duke said the painting had hung in a prominent position in the family home, Alnwick Castle, was well looked-after and had the artist's details on a name-plate.
The proceeds would go towards the upkeep of his collection of other artworks - reportedly worth more than £200m - and his estate, which was badly hit by the foot-and-mouth crisis, the duke wrote.
He added that he was "acting to invest in, and protect, a wider heritage".
Madonna of the Pinks, painted in the early 16th Century, is described as "one of the greatest old master paintings still in private hands in Britain" by the National Gallery.
A six-month export block is expected to be announced by arts minister Tessa Blackstone, and the gallery has applied for £30m of lottery money to fund the purchase.
Northumberland Estates owns 3,000 acres of land, with 200 tenant farmers and 300 employees.
It also runs two stately homes - Alnwick Castle in Northumberland and Syon House in London.
Madonna of the Pinks was bought by Algernon, the fourth Duke of Northumberland, in 1853.
Representatives of the National Gallery were not available for comment.