Over the last few decades, there has been a steady rise in the utilization of nanoparticles in the industry, agriculture, and health sectors. Nanoparticles have entered the food chain through air, soil, and water. However, lot of studies have been done since 2007 to explore the effect of the biological interaction of nanoparticles and its effect on drug delivery using animal models. Yet, the molecular mechanism of NP in biological systems is not clearly understood. The persistence of unstable NPs in the food web owing to decreased mobility is a major concern. However, green-synthesized nanomaterials are recognized as a relatively safer option. Therefore, in this study, both chemically and green synthesized AuNPs were synthesized, equilibrated, and compared for their colloidal stability and interaction with Brassica juncea leaf proteins. This study focuses on the comparative analysis of Chemically (Chem-AuNPs) and green synthesized gold nanoparticle (Green-AuNP) protein corona complex, specifically analyzing the differences between the soft and hard corona and their role in nanoparticle stability and homeostasis. They were incubated with Brassica juncea leaf crude for different time periods (1hr– 60hr), allowing proteins to adsorb onto the nanoparticle surface, forming a corona that influences cellular interactions, biodistribution, and physiology of plants. We characterized the protein composition in both the soft (loosely bound) and hard (tightly bound) corona using mass spectrometry, identifying key protein classes that mediate these interactions. Our findings suggest that certain protein groups, particularly those related to energy-yielding pathways bind to the corona proteins. Additionally, we observed distinct differences in protein composition between the soft and hard corona of both Chem- and Green-synthesized AuNPs, with the latter exhibiting more proteins that provide them thermodynamic stability. This work provides insights into the protein corona's role in modulating the activity of enzymes (Carbonic anhydrase, Malate dehydrogenase, and Fructose bis-phosphate aldolase) thus impacting the physiology of plants. The study also focuses on the interaction of AuNPs with RuBisCO that led to β-sheet domination in its secondary structure caused by α-helix to β- sheet transition.