The program of protein similarity searches, FASTP, was run on an IBM PC microcomputer to search proteins evolutionarily related to chymotrypsin. The compared chymotrypsin sequence was a bovine chymotrypsinogen A and the protein sequences searched in program were the 10,527 sequences in the National Biomedical Research Foundation library. Most of the proteins which were shown to be evolutionarily related to bovine chymotrypsinogen A were members of the serine protease family. As in the case of bovine carboxypeptidase A, however, some highly similar proteins to bovine chymotrypsinogen A were not serine proteases.
To get more informations on the evolution of chymotrypsin-related proteins, 7 proteins from the chymotrypsin-related proteins were multi-aligned by using a computer program, VAlign, which was run on a Cray 2S supercomputer. The result showed that there is an ancestral protein common to chymotrypsin-related proteins.
The molecular cloning of cDNA encoding bovine chymotrypsin was performed to study chymotrypsin at the level of gene. The bovine α-chymotrypsin purified by CM-cellulose column chromatography was used as immunogen to produce anti-chymotrypsin antibodies in rabbits. The anti-chymotrypsin antibodies were further purified by passing through Sepharose 4B column conjugated with bovine $₩alpha$- chymotrypsin. The affinity purified antibodies were used as probe in screening bovine pancreas cDNA library constructed in lambda gt11. In the first screening, several positively signaling plaques were isolated. But further identification of isolated plaques revealed that the signals of these plaques were not of true.
1차 아미노산 서열이 알려져 있는 단백질 은행 (National Biomedical Research Foundation protein sequence library)에서 소의 chymotrypsinogen A와 단백질 1차 구조상에서 높은 유사성을 보이는 단백질들을 조사하였다. 소의 chymotrypsinogen A와 유사성을 보인 단백질들은 몇몇 경우를 제외하곤 대부분 serine계 단백질 분해 효소였다. 소의 chymotrypsinogen A와 높은 유사성을 보인 6개의 단백질을 선택하여 1차 다중 정렬 배열을 실행하였다. 이 결과로부터 chymotrypsinogen A에 구조적으로 유사한 단백질들의 진화적 연관성을 분석할 수 있었다.
유전자 수준에서의 chymotrypsin 연구를 위해 소 chymotrypsin cDNA cloning을 시도하였다. 이온 교환 크로마토그래피법으로 순수분리한 소의 chymotrypsin을 토끼에 주사하여 혈청을 얻었다. 이 혈청을 친화 크로마토그래피로 정제하여 chymotrypsin에만 결합 특이성이 있는 항체를 얻어내었다. 정제된 항체를 probe로 하여 소의 췌장에서 분리된 mRNA 로부터 만들어진 cDNA library를 검색하였다. 1차 검색에서는 몇 개의 positive signal을 보이는 파아지를 얻어 내었다. 그러나 2차 검색과 DNA 분리 실험에서는 더이상 확인할 수 없었다.