Four nucleotides (A, U, C and G) constitute 64 codons at free combination but 64 codons are unequally assigned to 21 items (20 amino acids plus one stop). About 500 amino acids are known but only 20 are selected to make up the proteins. However, the relationships between amino acid and codon and between 20 amino acids have been unclear. In this paper, we studied the relationships between 20 amino acids in 33 species and found there were three constraints between 20 amino acids, such as the relatively stable mean carbon and hydrogen (C : H) ratios (0.50), similarity interactions between the constituent ratios of amino acids, and the frequency of amino acids according with Poisson distribution under certain conditions. We demonstrated that the unequal distribution of 64 codons and the choice of amino acids in molecular evolution would be constrained to remain stable C : H ratios. The constituent ratios and frequency of 20 amino acids in a species or a protein are two determinants of protein sequence evolution, so this finding showed the constraints between 20 amino acids played an important role in protein sequence evolution.

四种核苷酸（A、U、C 和 G）自由组合时构成 64 个密码子，但 64 个密码子不均等地分配给 21 个项目（20 个氨基酸加 1 个终止位点）。已知大约 500 种氨基酸，但仅选择 20 种来组成蛋白质。然而，氨基酸与密码子之间以及20个氨基酸之间的关系尚不清楚。在本文中，我们研究了33个物种的20个氨基酸之间的关系，发现20个氨基酸之间存在三个约束，例如相对稳定的平均碳氢（C：H）比（0.50）、成分之间的相似相互作用氨基酸的比例，以及在一定条件下氨基酸出现的频率符合泊松分布。我们证明了 64 个密码子的不均匀分布和分子进化中氨基酸的选择将受到限制以保持稳定的 C:H 比率。一个物种或一个蛋白质中20个氨基酸的组成比例和频率是蛋白质序列进化的两个决定因素，因此这一发现表明20个氨基酸之间的限制在蛋白质序列进化中发挥着重要作用。